Synthesis of the breast tumor-associated antigen defined by monoclonal antibody MBr1 and uses thereof

ABSTRACT

The present invention provides a process for synthesizing a compound having the structure: ##STR1## wherein R is H, as well as related oligosaccharides useful as a vaccine for inducing antibodies to human breast cancer cells in an adjuvant therapy therefor.

This invention was made with government support under grantsGM-15240-02, GM-16291-01, and AI-16943 from the National Institutes ofHealth. Accordingly, the U.S. Government has certain rights in theinvention.

This application is a divisional of U.S. Ser. No. 08/430,355, filed Apr.28, 1995, now U.S. Pat. No. 5,708,163, the contents of which are herebyincorporated by reference, which application is a continuation-in-partof U.S. Ser. No. 08/213,053, filed Mar. 15, 1994, now U.S. Pat. No.5,543,505, the contents of which are hereby incorporated by referenceinto this application.

BACKGROUND OF THE INVENTION

Throughout this application, citations for various publications areprovided within parentheses in the text. The disclosures of thesepublications are hereby incorporated in their entirety by reference intothis application in order to more fully describe the state of the art towhich this invention pertains.

The function of carbohydrates as structural materials and as energystorage units in biological systems is well recognized. By contrast, therole of carbohydrates as signaling molecules in the context ofbiological processes has only recently been appreciated. (M. L.Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S.Hakomori, J. C. Paulson, Science, 1990, 250, 1130; M. J. Polley, M. L.Phillips, E. Wagner, E. Nudelman, A. K. Singhal, S. Hakomori, J. C.Paulson, Proc. Natl. Acad. Sci. USA, 1991, 88, 6224: T. Taki, Y.Hirabayashi, H. Ishikawa, S. Kon, Y. Tanaka, M. Matsumoto, J. Biol.Chem., 1986, 261, 3075; Y. Hirabayashi, A. Hyogo, T. Nakao, K. Tsuchiya,Y. Suzuki, M. Matsumoto, K. Kon, S. Ando, ibid., 1990, 265, 8144; O.Hindsgaul, T. Norberg, J. Le Pendu, R. U. Lemieux, Carbohydr. Res.,1982, 109, 109; U. Spohr, R. U. Lemieux, ibid., 1988, 174, 211) Theelucidation of the scope of carbohydrate involvement in mediatingcellular interaction is an important area of inquiry in contemporarybiomedical research. The carbohydrate molecules, carrying detailedstructural information, tend to exist as glycoconjugates (cf.glycoproteins and glycolipids) rather than as free entities. Given thecomplexities often associated with isolating the conjugates inhomogeneous form and the difficulties in retrieving intact carbohydratesfrom these naturally occurring conjugates, the applicability ofsynthetic approaches is apparent. (For recent reviews of glycosylationsee: Paulsen, H., Agnew. Chemie Int. Ed. Engl., 1982, 21, 155; Schmidt,R. R., Angew. Chemie Int. Ed. Engl., 1986, 25, 212; Schmidt, R. R.,Comprehensive Organic Synthesis, Vol. 6, Chapter 1(2), Pergamon Press,Oxford, 1991; Schmidt, R. R., Carbohydrates, Synthetic Methods andApplications in Medicinal Chemistry, Part I, Chapter 4, VCH Publishers,Weinheim, New York, 1992. For the use of glycals as glycosyl donors inglycoside synthesis, see Lemieux, R. U., Can. J. Chem., 1964, 42, 1417;Lemieux, R. U., Faser-Reid, B., Can. J. Chem., 1965, 43, 1460; Lemieux,R. U., Morgan, A. R., Can. J. Chem., 1965, 43, 2190; Thiem, J., Karl,H., Schwentner, J., Synthesis, 1978, 696; Thiem. J. Ossowski, P.,Carbohydr. Chem., 1984, 3, 287; Thiem, J., Prahst, A., Wendt, T. LiebigsAnn. Chem., 1986, 1044; Thiem, J. in Trends in Synthetic CarbohydrateChemistry, Horton, D., Hawkins, L. D., McGarvvey, G. L., eds., ACSSymposium Series #386, American Chemical Society, Washington, D.C.,1989, Chapter 8.)

The carbohydrate domains of the blood group substances contained in bothglycoproteins and glycolipids are distributed in erythrocytes,epithelial cells and various secretions. The early focus on thesesystems centered on their central role in determining blood groupspecificities. (R. R. Race and R. Sanger, Blood Groups in Man, 6th ed.,Blackwell, Oxford, 1975) However, it is recognized that suchdeterminants are broadly implicated in cell adhesion and bindingphenomena. (For example, see M. L. Phillips, E. Nudelman, F. C. A.Gaeta, M. Perez, A. K. Singhal, S. Hakomori, J. C. Paulson, Science,1990, 250, 1130.) Moreover, ensembles related to the blood groupsubstances in conjugated form are encountered as markers for the onsetof various tumors. (K. O. Lloyd, Am. J. Clinical Path., 1987, 87, 129;K. O. Lloyd, Cancer Biol., 1991, 2, 421) Carbohydrate-based tumorantigenic factors might find applications at the diagnostic level, asresources in drug delivery or ideally in immunotherapy. (Toyokuni, T.,Dean, B., Cai, S., Boivin, D., Hakomori, S., and Singhal, A. K., J. Am.Chem Soc., 1994, 116, 395; Dranoff, G., Jaffee, E., Lazenby, A.,Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H.,Paardoll, D., Mulligan, R., Proc. Natl. Acad. Sci. USA, 1993, 90, 3539;Tao, M-H., Levy, R., Nature, 1993, 362, 755; Boon, T., Int. J. Cancer,1993, 54, 177; Livingston, P. O., Curr. Opin. Immunol., 1992, 4, 624;Hakomori, S., Annu. Rev. Immunol., 1984, 2, 103; K. Shigeta, et al., J.Biol. Chem., 1987, 262, 1358)

The present invention provides new strategies and protocols foroligosaccharide synthesis. The object is to simplify such constructionssuch that relatively complex domains can be assembled with highstereo-specifity. Major advances in glycoconjugate synthesis require theattainment of a high degree of convergence and relief from the burdensassociated with the manipulation of blocking groups. Another requirementis that of delivering the carbohydrate determinant with appropriateprovision for conjugation to carrier proteins or lipids. (Bernstein, M.A., and Hall, L. D., Carbohydr. Res., 1980, 78, Cl; Lemieux, R. U.,Chem. Soc. Rev., 1978, 7, 423; R. U. Lemieux, et al., J. Am. Chem. Soc.,1975, 97, 4076) This is a critical condition if the syntheticallyderived carbohydrates are to be incorporated into carriers suitable forbiological application.

Antigens which are selective or ideally specific for cancer cells couldprove useful in fostering active immunity. (Hakomori, S., Cancer Res.,1985, 45, 2405-2414; Feizi, T., Cancer Surveys, 1985, 4, 245-269) Novelcarbohydrate patterns are often presented by transformed cells as eithercell surface glycoproteins or as membrane-anchored glycolipids. Inprinciple, well chosen synthetic glycoconjugates which stimulateantibody production could confer active immunity against cancers whichpresent equivalent structure types on their cell surfaces. (Dennis, J.,Oxford Glycosystems Glyconews Second, 1992; Lloyd, K. O., in SpecificImmunotherapy of Cancer with Vaccines, 1993, New York Academy ofSciences pp. 50-58) Chances for successful therapy improve withincreasing restriction of the antigen to the target cell. Aglycosphingolipid was isolated by Hakomori and collaborators from thebreast cancer cell line MCF-7 and immunocharacterized by monoclonalantibody MBr1. (Bremer, E. G., et al., J. Biol. Chem., 1984, 259,14773-14777; Menard, S., et al., Cancer Res., 1983, 43, 1295-1300) Thenovel glycosphingolipid structure 1b (FIG. 8) was proposed for thisbreast tumor-associated antigen on the basis of methylation andenzymatic degradation protocols. A ¹ H NMR spectrum consistent with butnot definitive for the proposed structure was obtained from traceamounts of isolated antigen. While individual sectors of the proposedstructure were not unknown, the full structure was first described basedon studies on the breast cancer line. It should be noted that MBr1 alsobinds to normal human mammary gland tissue and ovarian cancer celllines. Therefore, 1b as a total entity is likely not restricted to thetransformed breast cells. Alternatively, smaller subsections of 1b areadequate for antibody recognition and binding. (The synthesis of the DEFfragment of 1b has been reported, and has been shown to bind to MBr1:Lay, L.; Nicotra, F.; Panza, L.; Russo, G. Helv. Chim. Acta, 1994, 77,509-514.)

The compounds prepared by processes described herein are antigens usefulin adjuvant therapies as a vaccines capable of inducing MBr1 antibodiesimmunoreactive with human breast tumor cells. Such adjuvant therapieshave potential to reduce the rate of recurrence of breast cancer andincrease survival rates after surgery. Clinical trials on 122 patentssurgically treated for AJCC stage III melanoma who were treated withvaccines prepared from melanoma differentiation antigen GM2 (anothertumor antigen which like MBr1 is a cell surface carbohydrate)demonstrated in patients lacking the antibody prior to immunization, ahighly significant increase in disease-free interval (P. O. Livingston,et al., J. Clin Oncol., 12, 1036 (1994)).

The present invention provides a method of synthesizing 1b in quantityas well as artificial protein-conjugates of the oligosaccharide whichmight be more immunogenic than the smaller glycolipid. The antigencontains a novel array of features including the α-linkage between the Band the C entities, as well as the β-linked ring D gal-NAc residue. (Forthe synthesis of a related structure (SSEA-3) which lacks the fucoseresidue see: Nunomura, S.; Ogawa, T., Tetrahedron Lett., 1988, 29,5681-5684.) The present invention provides (i) a total synthesis of 1b,(ii) rigorous proof that the Hakomori antigen does, in fact, correspondto 1b and (iii) the synthesis of a bioconjugatable version of 1b. Theconciseness of the synthesis reflects the efficiency of glycal assemblymethods augmented by a powerful method for sulfonamidoglycosylation(see, e.g., the transformation of 14b-15b, FIG. 10).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows glycal assembly leading to neoglycoproteins.

FIG. 2 shows the synthesis of 4a. Reagents: a) TBDPSCL, imidazole/DMF84%; b) carbonyldiimidazole, cat. imidazole, THF (65%) c) 5a,di-tert-butylpyridine, AgClO₄, SnCl₂, ether (51%); PhSO₂ NH₂,1(sym-coll)₂ ClO₄ (94%).

FIG. 3 shows the synthesis of 8a. Reagents: a) 9a, AgBF₄, 4A mol.sieves, THF (75%); b) i. TBAF, THF; ii. Na/NH₃ ; iii Ac₂ O, pyr. c) i.3,3-dimethyldioxirane; allyl alcohol, ZnCl₂ (72%); ii. NaOMe, MeOH(quant.).

FIG. 4 shows a strategy for the solid-phase synthesis ofoligosaccharides using the glycal assembly method.

FIG. 5 shows the application of the solid-support method to the assemblyof 1,2-branching patterns of complex carbohydrates.

FIG. 6 shows the synthesis of a tetrasaccharide having H-type 2 bloodgroup specificity. Reagents: (a) 1. 3,3-dimethyldioxirane, CH₂ Cl₂ ; 2.8, ZnCl₂, THF; (b) 10, Sn(OTf)₂, di-tert-butylpyridine, THF; (c) TBAF,AcOH, THF; (d) TIPSCl, imidazole, DMF; (e) I(coll)₂ ClO₄, PhSO₂ NH₂, CH₂Cl₂ ; (f) 15, AgBF₄, 4A M.S., THF; (g) 1. TBAF, AcOH, THF; 2. Na/NH₃ ;3. Ac₂ O, pyridine.

FIGS. 7a and 7b show the synthesis of a Le^(b) hexasaccharide inbioconjugatable form. Reagents: (a) 1. 3,3-dimethyldioxirane, CH₂ Cl₂ ;2. 19, ZnCl₂, THF; (b) 10, Sn(OTf)₂ di-tert-butylpyridine, THF; (c)TBAF, AcOH, THF; (d) TIPSCl, imidazole, DMF; (e) I(coll)₂ ClO₄, PhSO₂NH₂, CH₂ Cl₂ ; (f) 24, AgBF₄, 4A M.S., THF; (g) 1. TBAF, AcOH, THF; 2.Na/NH₃ ; 3. Ac₂ O, pyridine; (h) 1. 3,3-dimethyldioxirane, CH₂ Cl₂ ; 2.allyl alcohol, ZnCl₂ ; 3. NaOMe, MeOH.

FIG. 8 shows the structure of the MBr1 antigen and a reaction pathway toa trisaccharide intermediate.

Reagents: a. n-Bu₂ SnO, PMBCl, TBABr, PhH, 70%; b. NaH, BnBr, DMF, 95%;c. (i) 3,3-dimethyldioxirane, CH₂ Cl₂ ; (ii) TBAF, THF; (iii) NaH, BnBr,DMF, 40% (three steps); d. NaH, BnBr, DMF, 80%; e. (i) TBAF, THF; (ii)NaOMe, MeOH, 93% (two steps); f. (n-Bu₃ Sn) ₂ O, BnBr, TBABr, PhH,90%;g. SnCl₂, AgClO₄, 2,6-di-butylpyridine, 4 Å mol. sieves, Et₂ O, 40%α (4.5:1 α:B); h. DDQ, CH₂ Cl₂, H₂ O, 84%.

FIG. 9 shows a reaction pathway to a trisaccharide intermediate.

Reagents: a. (i) 3,3-dimethyldioxirane, CH₂ Cl₂ ; (ii) 10a, ZnCl₂, THF,87%; b. SnCl₂, AgClO₄, Et₂ O, 47%; c. I(coll)₂ ClO₄, PhSO₂ NH₂, 4 Å mol.sieves, 47%.

FIG. 10(a) shows a reaction pathway to the hexasaccharide MBr1 antigen.

Reagents: a. EtSH, LiHMDS, DMF, 75%. B. 8b (0.5 equiv), MeOTf, 4 Å Mol.sieves, 70-85% B, (10:1 B α); c. (i) 3,3-dimethyldioxirane, CH₂ Cl₂ (ii)17b (5 equiv), Zn(OTf)₂, 20%;.d. Ac₂ O, Et₃ N, DMAP, CH₂ Cl₂ 95%; e.Lindlar's cat., H₂ palmitic anhydride, EtOAc, 90%; f. (i) TBAF, THF;(ii) NaOMe, MeOH, 94%; g. (i) Na, NH₃, THF; (ii) Ac₂ O, Et₃ N, DMAP, CH₂Cl₂, 80% h. NaOMe, MeOH, quant.

FIG. 10(b) shows a reaction pathway to the allyl glycoside.

Reagents: a. TBAF, THF, 94%; b. (i) Na, NH3, THF; (ii) Ac₂ O, Et₃ N,DMAP, THF, DMF, 85%; c. (i) 3,3-dimethyldioxirane, CH₂ cl₂, (ii) allylalcohol, 65% (+29% of α-manno isomer); d. NaOMe, MeOH, quant.

FIG. 11 shows a reaction pathway to intermediates for reparing thehexasaccharide antigen MBr1.

FIG. 12 shows a reaction pathway to the hexasaccharide antigen MBr1 by a4+2 synthetic approach.

SUMMARY OF THE INVENTION

The present invention provides a process for synthesizing a compoundhaving the structure: ##STR2## wherein R is H.

The present invention also provides a process for synthesizing atrisaccharide ceramide having the structure: ##STR3##

The present invention further provides a process for synthesizing amercaptotrisaccharide having the structure: ##STR4##

The present invention also provides a process of synthesizing ahexasaccharide ceramide having the structure: ##STR5##

The present invention also provides a process of synthesizing ahexasaccharide ceramide having the structure: ##STR6##

The present invention also provides a process of synthesizing an allylhexasaccharide having the structure: ##STR7##

The present invention provides a process of synthesizing ahexasaccharide having the structure: ##STR8##

The present invention further provides a compound having the structure:##STR9## wherein n is an integer between about 0 and about 9.

The present invention also provides a compound having the structure:##STR10## wherein n is an integer between about 0 and about 9.

The present invention also provides a compound having the structure:##STR11## wherein n is an nteger between about 0 and about 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the structure:##STR12## wherein A is selected from the group consisting of (i) anamino acid bearing an ω-amino group or an ω-(C═O)-- group, (ii) an aminoacid residue of a peptide, which residue bears an ω-amino group or anω-(C═O)-- group, and (iii) an amino acid residue of a protein, whichresidue bears an ω-amino group or an ω-(C═O)-- group; wherein R₁ is H,OH, NH₂ or NHR₄, where R₄ is SO₂ Ph, a linear or branched chain alkyl oracyl group, or an aryl group; wherein M has the structure: ##STR13##wherein n is an integer from 0 to 18, and where n is greater than 1,each M is independently the same or different; wherein p is either 0 or1; wherein R₂, R₃, R₅ and R₆ are independently the same or different andare H or OH, with the proviso that geminal R₂ and R₃ are not both OH,and geminal R₅ and R₆ are not both OH; wherein each wavy line between acarbon atom and an oxygen atom denotes an R or S configuration at thecarbon atom; wherein X and Y are independently the same or different andare H₂ or O; and wherein k is an integer greater than or equal to 1,with the proviso that when A is an amino acid bearing an ω-amino groupor an ω-(C═O)-- group, k is equal to 1.

In one embodiment, the present invention provides the compound disclosedhereinabove wherein A is lysine or a lysine residue.

In another embodiment, the present invention provides the compounddisclosed hereinabove wherein A is glutamic acid or a glutamic acidresidue.

In another embodiment, the present invention provides the compounddisclosed hereinabove wherein A is aspartic acid or an aspartic acidresidue.

The invention also provides the compound disclosed hereinabove wherein Ais an amino acid residue of a globular protein. In one embodiment, theinvention provides the compound wherein the globular protein is selectedfrom the group consisting of bovine serum albumin and human serumalbumin.

In one embodiment, the invention provides the compound disclosedhereinabove wherein k is 1.

In another embodiment, the invention provides the compound disclosedhereinabove wherein n and p are both equal to 0.

The invention provides a compound having the structure: ##STR14##wherein R₁ is H, OH, NH₂ or NHR₄, where R₄ is SO₂ Ph, a linear orbranched chain alkyl or acyl group, or an aryl group; wherein M has thestructure: ##STR15## wherein n is an integer from 0 to 18, and where nis greater than 1, each M is independently the same or different;wherein R₂, R₃, R₅ and R₆ are independently the same or different andare H or OH, with the proviso that geminal R₂ and R₃ are not both OH,and geminal R₅ and R₆ are not both OH; wherein each wavy line between acarbon atom and an oxygen atom denotes an R or S configuration at thecarbon atom; and wherein R₇ is a substituted or unsubstituted allylgroup.

The invention also provides a compound having the structure: ##STR16##wherein n is an integer from 1 to 18; wherein R is H or a linear orbranched chain acyl group; wherein R₁ is H, OH, NH₂ or NHR₄, where R₄ isSO₂ Ph, a linear or branched chain alkyl or acyl group, or an arylgroup; and wherein R₂ is a substituted or unsubstituted allyl group. Inone embodiment, the invention provides the compound wherein n is 1.

The invention further provides a compound having the structure:##STR17## wherein R is H or a linear or branched chain acyl group;wherein R₁ is H, OH, NH₂ or NHR₄, where R₄ is SO₂ Ph, a linear orbranched chain alkyl or acyl group, or an aryl group; and wherein R₂ isa substituted or unsubstituted allyl group.

The invention also provides a compound having the structure: ##STR18##wherein R is H or a linear or branched chain acyl group; wherein R₁ isH, OH, NH₂ or NHR₄, where R₄ is SO₂ Ph, a linear or branched chain alkylor acyl group, or an aryl group; wherein R₂ is a substituted orunsubstituted allyl group; and wherein n is an integer from 1 to 18. Inone embodiment, the invention provides the compound wherein n is 1.

The invention also provides a compound having the structure: ##STR19##wherein R is H or a linear or branched chain acyl group.

The invention also provides a process for synthesizing a compound havingthe structure: ##STR20## wherein R is a substituted or unsubstitutedallyl group, which comprises the steps of (a) synthesizing a compoundhaving the structure: ##STR21## wherein R is a trialkylsilyl,aryldialkylsilyl, alkyldiarylsilyl or triarylsilyl group; (b) reactingthe compound of step (a) with a compound having structure: ##STR22##under suitable conditions to form a compound having the structure:##STR23## wherein R is a trialkylsilyl, aryldialkylsilyl,alkyldiarylsilyl or triarylsilyl group; (c) reacting the compound formedin step (b) with a compound having the structure: ##STR24## undersuitable conditions to form a compound having the structure: ##STR25##wherein R is a trialkylsilyl, aryldialkylsilyl, alkyldiarylsilyl ortriarylsilyl group; (d) deprotecting and re-protecting the compoundformed in step (c) under suitable conditions to form a compound havingthe structure: ##STR26## wherein R is TIPS; (e) iodosulfonamidating thecompound formed in step (d) under suitable conditions to form a compoundhaving the structure: ##STR27## (f) reacting the compound formed in step(e) with a compound having the structure: ##STR28## under suitableconditions to form a compound having the structure: ##STR29## wherein Ris H; (g) deprotecting and peracetylating the compound formed in step(f) under suitable conditions to form a compound having the structure:##STR30## (h) epoxidizing the compound formed n step (g) under suitableconditions to form an epoxide thereof and reacting the epoxide undersuitable conditions to form a compound having the structure: ##STR31##wherein R is a substituted or unsubstituted allyl group; and (i)treating the compound formed in step (h) under suitable conditions toform a compound having the structure: ##STR32## wherein R is asubstituted or unsubstituted allyl group. In the above process thesuitable conditions necessary for the various reactions and treatmentsmay be found in the Experimental Details section which followshereinafter. However, it is within the confines of the present inventionthat the specific reagents and solvents provided as well as the specificconditions necessary for reaction or treatment may be substituted withother suitable reactants, solvents and conditions well known to thoseskilled in the art.

The allyl compound may be conjugated to a peptide or protein via amineor carboxylic acid side chain. In practicing the invention, abioconjugate is prepared according to the protocol of Bernstein and Hall(Carbohydr. Res. 1980, 78, C1). The allyl group is ozonolyzed to formeither an aldehyde or carboxylic acid, which is condensed to a terminalamine to form, respectively, an imine or an amide. The imine is reducedwith sodium borohydride to the amine. Alternatively, the aldehyde isreductively aminated using procedures known in the art to form an aminewhich is reacted with a sidechain terminal carboxylic acid to form anamide conjugate.

The invention provides a pharmaceutical composition which comprises atherapeutically effective amount of the compound disclosed hereinaboveand a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.01-0.1M and preferably0.05M phosphate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preserva-tives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases and the like.

The invention further provides a method for treating a subject afflictedwith a disorder caused by Helicobacter pylori which comprisesadministering to the subject a therapeutically effective amount of thepharmaceutical composition disclosed hereinabove so as to treat thesubject afflicted with the disorder.

In one embodiment, the invention provides a method of treating a subjectafflicted with gastric or duodenal ulcer.

In another embodiment, the invention provides a method of treating asubject afflicted with gastric adenocarcinoma.

In addition, the invention provides a method for inhibiting the adhesionof Helicobacter pylori to gastric epithelium in a subject whichcomprises administering to the subject an amount of the compounddisclosed hereinabove effective to inhibit the adhesion of Helicobacterpylori to gastric epithelium in the subject.

The present invention also provides a process for synthesizing acompound having the structure: ##STR33## wherein R is H which comprises:(a)(i) reacting a compound having the structure: ##STR34## with anepoxidizing agent under suitable conditions to form an epoxide; (ii)cleaving the epoxide formed in step (a)(i) under suitable conditionswith RNF wherein each R is independently the same or different and is alinear or branched chain alkyl, aralkyl or aryl group to form afluoroalcohol; and (iii) alkylating the fluoroalcohol formed in step(b)(ii) under suitable conditions with a non-nucleophilic base and anorganic halide having the formula C₆ H₅ CH₂ X wherein X is Br, Cl, I orF to form a compound having the structure: ##STR35## (b)(i) synthesizinga compound having the structure: ##STR36## (c)(i) treating the compoundformed in step (b) with an epoxidizing agent under suitable conditionsto form an epoxide; and (ii) coupling the epoxide formed in step (c)(i)with a compound having the structure: ##STR37## under suitableconditions to form a compound having the structure: ##STR38## (d)(i)alkylating the compound formed in step (c)(ii) under suitable conditionswith a non-nucleophilic base and an organic halide having the formula C₆H₅ CH₂ X wherein X is Br, Cl, I or F; and (ii) de-silylating thecompound formed in step (d)(i) under suitable conditions with R₄ NFwherein each R is independently the same or different and is a linear orbranched chain alkyl, aralkyl or aryl group; (iii) treating the compoundformed in step (d)(ii) under suitable conditions with a metal alkoxideto form a deprotected disaccharide; and (iv) alkylating the disaccharideformed in step (d)(iii) under suitable conditions to form a selectivelydeprotected disaccharide having the structure: ##STR39## (e)(i) couplingthe selectively deprotected disaccharide formed in step (d)(iv) with thecompound formed in step (a)(iii) under suitable conditions to form aprotected trisaccharide; and (ii) de-protecting the protectedtrisaccharide formed in step (e)(i) under suitable conditions to form atrisaccharide having the structure: ##STR40## wherein R is H. In step(a) reaction (i) may be carried out using a variety of epoxidizingagents including peracetic acid, m-chloroperbenzoic acid,trifluoroperacetic acid, and hydrogen peroxide. A preferred agent is3,3-dimethyldioxirane. Non-nucleophilic, inert solvents may be used,such as dichloromethane. Reaction (a)(ii) may be performed using organicammonium fluoride salts, including tetrabutylammonium fluoride, in arange of solvents, including ethereal solvents, preferably intetrahydrofuran. Reaction (a)(iii) may be performed using anon-nucleophilic base such as sodium hydride in a non-nucleophilicsolvent such as DMF. In step (b) the compound shown may be prepared asdescribed herein. Step (c)(i) may be carried out using a variety ofepoxidizing agents including peracetic acid, m-chlorobenzoic acid,trifluoroacetic acid, and hydrogen peroxide, 3,3-dimethyldioxirane beingpreferred, in non-nucleophilic, inert solvents, such as dichloromethane.Coupling step (c)(ii) may be carried out using a metal catalyst, such aszinc chloride, in an inert solvent, such as THF. Step (d)(i) is carriedout using a non-nucleophilic base such as sodium hydride in anon-nucleophilic solvent such as DMF. In step (d)(ii) de-silylation iseffected using an organic ammonium fluoride salt, includingtetrabutylammonium fluoride, in a range of solvents, including etherealsolvents, preferably in tetrahydrofuran. The carbonate ester is cleavedusing a metal alkoxide, such as sodium methoxide, in an alcoholic mediumsuch as methanol. Step (d)(iv) is selectively performed using a metaloxide, such as (n-Bu₃ Sn)₂ O, in the presence of an organic ammoniumbromide, such as tetra-n-butylammonium bromide, in an inert solvent suchas benzene. Step (e) is a coupling performed in the presence of a metalhalide salt, such as SnCl₂, in the presence of silver perchlorate and2,6-di-t-butylpyridine, in a solvent, such as ether, containingmolecular sieves. Oxidative removal of PMB is performed with anoxidizing agent such as DDQ in an inert solvent system, which maypreferably be heterogeneous, for example, using water/dichloromethane.

The present invention also provides a process for synthesizing atrisaccharide ceramide having the structure: ##STR41## which comprises:(a) synthesizing a trisaccharide having the structure: ##STR42## whereinR is PMB; (b)(i) reacting the trisaccharide formed in step (a) with anepoxidizing agent under suitable conditions to form a trisaccharideepoxide; and (ii) reacting the trisaccharide epoxide formed in step(b)(i) with a compound having the structure: ##STR43## under suitableconditions to form a protected trisaccharide ceramide having thestructure: ##STR44## (c)(i) acylating the ceramide formed in step(b)(ii) under suitable conditions; and (ii) selectively de-protectingthe compound formed in step (c)(i) under suitable conditions to form thetrisaccharide ceramide.

In step (a) the trisaccharide may be synthesized as described herein.Step (b)(i) is performed using using a variety of epoxidizing agentsincluding peracetic acid, m-chlorobenzoic acid, trifluoroacetic acid,and hydrogen peroxide, 3,3-dimethyldioxirane being preferred, innon-nucleophilic, inert solvents, such as dichloromethane. Coupling step(b)(ii) may be carried out using a tributyltin ether of the ceramideprecursor and a metal catalyst, such as zinc chloride, in an inertsolvent, such as THF. In step (c)(i) acylation is performed using alinear or branched chain alkyl anhydride preferably acetic anhydride orhalide in the presence of triethylamine and DMAP in an inert organicsolvent such as dichloromethane. The PMB protecting group is removedoxidatively, preferably as described above.

The present invention further provides a process for synthesizing amercaptotrisaccharide having the structure: ##STR45## which comprises:(a)(i) synthesizing a compound having the structure: ##STR46## (ii)coupling the compound of step (a)(i) with a compound having structure:##STR47## under suitable conditions to form a disaccharide having thestructure: ##STR48## (b) coupling the disaccharide formed in step(a)(ii) with a compound having the structure: ##STR49## under suitableconditions to form a trisaccharide having the structure: ##STR50## (c)iodosulfonamidating the trisaccharide formed in step (b) under suitableconditions to form a iodosulfonamide having the structure: ##STR51## and(d) reacting the iodosulfonamide formed in step (c) under suitableconditions with a thiolate to form the mercaptotrisaccharide.

Step (a)(ii) is performed by reacting the compound of step (a)(i), whichmay be obtained as described herein or otherwise, with a variety ofepoxidizing agents including peracetic acid, m-chloroperbenzoic acid,trifluoroperacetic acid, and hydrogen peroxide, 3,3-dimethyldioxiranebeing preferred, in non-nucleophilic, inert solvents, such asdichloromethane, followed by coupling with the diol monosaccharide ofstep (a)(ii) which may be carried out using a metal catalyst, such aszinc chloride, in an inert solvent, such as THF. Coupling with thefluorosugar is carried out in step (b) in the presence of a metal halidesalt, such as SnCl₂, in the presence of silver perchlorate and2,6-di-t-butylpyridine, in a solvent, such as ether, containingmolecular sieves. Step (c) is performed using I(coll)₂ perchlorate andPhSO₂ NH₂ in the presence of molecular sieves. Step (d) is carried outusing alkyl thiol and a base such as LiHMDS in an inert solvent as DMF.

The present invention also provides a process of synthesizing ahexasaccharide ceramide having the structure: ##STR52## which comprises:a) coupling a compound having the structure: ##STR53## with a compoundhaving the structure: ##STR54## under suitable conditions to form acompound having the structure: ##STR55## (b)(i) reacting the compoundformed in step (a) with an epoxidizing agent under suitable conditionsto form a hexasaccharide epoxide; and (ii) reacting the hexasaccharideepoxide with a stannyl ether having the structure: ##STR56## undersuitable conditions to form a hexasaccharide alcohol; (c) acylating thehexasaccharide alcohol formed in step (b)(ii) under suitable conditionsto form a hexasaccharide acetate having the structure: ##STR57## (d)reductively acylating the hexasaccharide acetate formed in step (c)under suitable conditions in the presence of palmitic anhydride to forma hexasaccharide ceramide; (e) desilylating and partially deprotectingthe hexasaccharide ceramide under suitable conditions to form apartially deprotected hexasaccharide ceramide; (f) (i) reducing thepartially deprotected hexasaccharide ceramide under suitable conditionsto form a deprotected hexasaccharide ceramide acetate; and (ii)acylating the deprotected hexasaccharide ceramide acetate under suitableconditions to form a hexasaccharide ceramide peracetate; and (g)saponifying the hexasaccharide ceramide peracetate under suitableconditions to form the hexasaccharide ceramide.

Step (a) is performed using triflate esters, such as methyl triflate, inthe presence of molecular sieves in an inert solvent. Step (b)(i) iscarried out using a variety of epoxidizing agents including peraceticacid, m-chloroperbenzoic acid, trifluoroperacetic acid, and hydrogenperoxide, 3,3-dimethyldioxirane being preferred, in non-nucleophilic,inert solvents, such as dichloromethane. Step (b)(ii) is performed usinga stannyl ether of the ceramide precursor, preferably the tri-n-butylstannylether, in the presence of a metal salt, such as Zn triflate, inan inert solvent, such as THF. Step (c) is carried out using aceticanhydride in the presence of a base such as triethylamine and DMAP. Step(d) is carried out using a noble metal catalyst such as Lindlar'scatalyst and hydrogen gas in the presence of palmitic anhydride in aninert solvent such as ethyl acetate. Desilylation step (e) is effectedusing organic ammonium fluoride salts, such as tetra-n-butylammoniumfluoride in THF. The carbonate ester is cleaved using a metal alkoxidesuch as NaOMe in an alcohol such as methanol. In step (f)(i) reductionis performed using a metal such as lithium or sodium in liquid ammoniaand an inert solvent such as THF. Step (f)(ii) is carried out usingacetic anhydride in the presence of a base such as Et₃ N and DMAP in aninert solvent such as dichloromethane. The peracetate is saponifiedusing a metal alkoxide such as sodium methoxide in an alcohol such asmethanol.

The present invention also provides a process of synthesizing ahexasaccharide ceramide having the structure: ##STR58## which comprises:(a) coupling a compound having the structure: ##STR59## with a compoundhaving the structure: ##STR60## under suitable conditions to form ahexasaccharide having the structure: ##STR61## and (b)(i) reducing thehexasaccharide formed in step (a) under suitable conditions in thepresence of palmitic anhydride to form a palmitoyl amide; (ii)desilylating the palmitoyl amide with R₄ NF wherein each R isindependently the same or different and is a linear or branched chainalkyl, aralkyl or aryl group under suitable conditions to form apartially deprotected hexasaccharide; (iii) de-protecting thehexasaccharide formed in step (b)(ii) under suitable conditions to forma deprotected hexasaccharide; (iv) acylating the hexasaccharide formedin step (b)(iii) under suitable conditions to form a hexasaccharideceramide peracetate; and (v) saponifying the hexasaccharide ceramideperacetate under suitable conditions to form the hexasaccharideceramide.

Step (a) is performed using triflate esters, such as methyl triflate, inthe presence of molecular sieves in an inert solvent. Step (b)(i) iscarried out using using a noble metal catalyst such as Lindlar'scatalyst and hydrogen gas in the presence of palmitic anhydride in aninert solvent such as ethyl acetate. Step (b)(ii) is performed usingorganic ammonium fluoride salts, such as tetra-n-butylammonium fluoridein THF. In step (b)(iii) reduction is performed using a metal such aslithium or sodium in liquid ammonia and an inert solvent such as THF.Step (b)(iv) is carried out using acetic anhydride in the presence of abase such as Et₃ N and DMAP in an inert solvent such as dichloromethane.In step (v) the peracetate carbonate is saponified using a metalalkoxide such as sodium methoxide in an alcohol such as methanol.

The present invention also provides a process of synthesizing an allylhexasaccharide having the structure: ##STR62## which comprises: (a)coupling a compound having the structure: ##STR63## with a compoundhaving the structure: ##STR64## wherein R is H under suitable conditionsto form a hexasaccharide having the structure: ##STR65## (b)(i)desilylating the compound formed in step (a) with R₄ NF wherein each Ris independently the same or different and is a linear or branched chainalkyl, aralkyl or aryl group under suitable conditions to form apartially deprotected hexasaccharide; (ii) de-protecting thehexasaccharide formed in step (b)(i) under suitable conditions to form adeprotected hexasaccharide; and (iii) peracylating the compound formedin step (b)(ii) under suitable conditions to form a hexasaccharideperacetate having the structure: ##STR66## (c)(i) reacting thehexasaccharide peracetate formed in step b)(iii) with an epoxidizingagent under suitable conditions to form an hexasaccharide epoxideperacetate; (ii) treating the hexasaccharide epoxide peracetate formedin step (c)(i) with allyl alcohol under suitable conditions to form anallyl hexasaccharide peracetate; and (iii) saponifying the allylhexasaccharide peracetate under suitable conditions to form the allylhexasaccharide.

Step (a) is performed using triflate esters, such as methyl triflate, inthe presence of molecular sieves in an inert solvent. Step (b)(i) iscarried out using organic ammonium fluoride salts, such astetra-n-butylammonium fluoride in THF. Step (b)(i) is performed using ametal alkoxide such as sodium methoxide in an alcohol such as methanol,followed by reduction performed using a metal such as lithium orpreferably sodium in liquid ammonia and an inert solvent such as THF.Step (b)(iii) is carried out using acetic anhydride in the presence of abase such as Et₃ N and DMAP in an inert solvent such as dichloromethane.In step (c)(i) is carried out using a variety of epoxidizing agentsincluding peracetic acid, m-chlorobenzoic acid, trifluoroacetic acid,and hydrogen peroxide, 3,3-dimethyldioxirane being preferred, innon-nucleophilic, inert solvents, such as dichloromethane. Step (c)(ii)is carried out using allyl alcohol in an inert solvent. Step (c)(iii)the peracetate carbonate is saponified using a metal alkoxide such assodium methoxide in an alcohol such as methanol.

The present invention provides a process of synthesizing ahexasaccharide having the structure: ##STR67## which composes: (a)coupling a compound having the structure: ##STR68## with a compoundhaving the structure: ##STR69## under suitable conditions to form acompound having the structure: ##STR70## (b)(i) acylatinq the compoundformed in step (a) under suitable conditions; and (ii) reacting thecompound formed in step (b)(i) with an epoxidizing agent under suitableconditions to form an epoxide having the structure: ##STR71## (c)(i)treating the epoxide with R₄ NF wherein each R is independently the sameor different and is a linear or branched chain alkyl, aralkyl or arylgroup under suitable conditions; and (ii) alkylating the compound formedin step (c)(i) under suitable conditions to form a compound having thestructure: ##STR72## wherein R is H or acyl; (d) coupling the compoundformed in step (c)(ii) with a compound having the structure: ##STR73##under suitable conditions to form the hexasaccharide.

Step (a) is performed using a metal catalyst such as silvertetrafluooborate in an inert solvent. Step (b) is carrned out usingacetic anhydride in the presence of a base such as Et₃ N and DMAP in aninert solvent such as dichloromethane. Step (b)(ii) is carried out usinga variety of epoxiddizing agents including peracetic acid,m-chiorobenzoic acid, trifluoroacetic acid, and hydrogen peroxide,3,3-dimethyldioxirane being preferred, in non-nucleophilic, inertsolvents, such as dichloromethane. Step (c)(i) is effected with organicammonium fluoride salts, such as tetra-n-butylammonium fluoride in THF.Step (c)(ii) is performed using a non-nucleophilic base such as sodiumhydride in an inert solve. Step (d) is performed using a metal saltcatalyst such as tin dichloride in the presence of silver perchlorate inan inert solvent such as di-t-butylpyridine. Further transformationsprovide deprotected products or conjugates with proteins or othercarriers.

The present invention further provides a compound having the structure:##STR74## wherein n is an integer between about 0 and about 9. The allylglycoside shown is prepared using the glycal coupling methods taughtherein, and may be bound to protein carriers using general reactionsdescribed herein or by standard methods in the art. For example, theallyl glycoside may be prepared by coupling compound 9b disclosed hereinwith a suitably protected 8b, followed by coupling with 12b, thencoupling with allyl alcohol and an appropriate deprotection sequence.

The present invention also provides a compound having the structure:##STR75## wherein n is an integer between about 0 and about 9.

The allyl glycoside shown is prepared using the glycal coupling methods,allylation and a deprotection sequence as taught herein (see FIG. 12),and may be bound to protein carriers using general reactions describedherein or by standard methods in the art.

The present invention also provides a compound having the structure:##STR76## wherein n is an integer between about 0 and about 9.

The allyl glycosides shown are prepared using the glycal couplingmethods taught herein, and may be bound to protein carriers usinggeneral reactions described herein or by standard methods in the art.

It is within the scope of the present invention to vary the combinationof protecting groups for the various sugar hydroxyl groups in accordwith ordinary skill in the art.

The present invention provides a method of inducing antibodies in ahuman subject, wherein the antibodies are immunoreactive with humanbreast tumor cells, which comprises administering to the subject anamount of a compound having the structure: ##STR77## alone or bound to asuitable immunological adjuvant effective to induce the antibodies. Inone embodiment, the present invention provides a method wherein theantibodies induced are MBr1 antibodies. In another embodiment, thepresent invention provides a method wherein the subject is in clinicalremission or, where the subject has been treated by surgery, has limitedunresected disease. In another embodiment, the present inventionprovides a method wherein the adjuvant is a protein carrier, bacteria orliposomes. In yet another embodiment, the present invention provideswherein the adjuvant is bacille Calmette-Guerin (BCG).

The present invention provides a method of preventing recurrence ofbreast cancer in a subject which comprises vaccinating the subject withthe compound shown hereinabove either alone or bound to a suitableimmunological carrier, adjuvant or vehicle.

The present invention also provides a method of inducing antibodies in asubject, wherein the antibodies are immunoreactive with human breasttumor cells, which comprises administering to the subject an amount ofthe compound having the structure: ##STR78## wherein n is an integerbetween about 0 and about 9 either alone or bound to a suitableimmunological adjuvant effective to induce the antibodies. In oneembodiment, the present invention provides a method wherein theantibodies induced are MBr1 antibodies. In another embodiment, thepresent invention provides a method wherein the subject is in clinicalremission or, where the subject has been treated by surgery, has limitedunresecced disease. In another embodiment, the present inventionprovides a method wherein the adjuvant is a protein carrier, bacteria orliposomes. In yet another embodiment, the present invention provides amethod wherein the adjuvant is bacille Calmette-Guerin.

The present invention provides a method of preventing recurrence ofbreast cancer in a subject which comprises vaccinating the subject withthe compound shown hereinabove either alone or bound to a suitableimmunological carrier, adjuvant or vehicle.

The present invention also provides a method of inducing antibodies in asubject, wherein the antibodies are immunoreactive with human breasttumor cells, which comprises administering to the subject an amount ofthe compound having the structure: ##STR79## wherein n is an integerbetween about 0 and about 9 either alone or bound to a suitableimmunological adjuvant effective to induce the antibodies. In oneembodiment, the present invention provides a method wherein theantibodies induced are MBr1 antibodies. In another embodiment, thepresent invention provides a method wherein the subject is in clinicalremission or, where the subject has been treated by surgery, has limitedunresected disease. In another embodiment, the present inventionprovides a method wherein the adjuvant is a protein carrier, bacteria orliposomes. In yet another embodiment, the present invention provides amethod wherein the adjuvant is bacille Calmette-Guerin.

The present invention also provides a method of preventing recurrence ofbreast cancer in a subject which comprises vaccinating the subject withthe compound shown hereinabove either alone or bound to a suitableimmunological carrier, adjuvant or vehicle.

The present invention additionally provides a method of inducingantibodies in a subject, wherein the antibldies are immunoreactive withhuman breast tumor cells, which comprises administering to the subjectan amount of the compound having the structure: ##STR80## wherein n isan integer between about 0 and about 9 either alone or bound to asuitable immunological adjuvant effective to induce the antibodies. Inone embodiment, the present invention provides a method wherein theantibodies induced are MBr1 antibodies. In another embodiment, thepresent invention provides a method wherein the subject is in clinicalremission or, where the subject has been treated by surgery, has limitedunresected disease. In another embodiment, the present inventionprovides a method wherein the adjuvant is a protein carrier, bacteria orliposomes. In yet another embodiment, the present invention provideswherein the adjuvant is bacille Calmette-Guerin.

The present invention also provides a method of preventing recurrence ofbreast cancer in a subject which comprises vaccinating the subject withthe compound shown hereinabove either alone or bound to a suitableimmunological carrier, adjuvant or vehicle.

EXPERIMENTAL DETAILS

General Procedures

All air- and moisture-sensitive reactions were performed in aflame-dried apparatus under an argon atmosphere unless otherwise noted.Air-sensitive liquids and solutions were transferred via syringe orcanula. Wherever possible, reactions were monitored by thin-layerchromatography (TLC). Gross solvent removal was performed in vacuumunder aspirator vacuum on a Buchi rotary evaporator, and trace solventwas removed on a high vacuum pump at 0.1-0.5 mmHg.

Melting points (mp) were uncorrected and performed in soft glasscapillary tubes using an Electrothermal series IA9100 digital meltingpoint apparatus.

Infrared spectra (IR) were recorded using a Perkin-Elmer 1600 seriesFourier-Transform instrument. Samples were prepared as neat films onNaCl plates unless otherwise noted. Absorption bands are reported inwavenumbers (cm⁻¹).

Only relevant, assignable bands are reported.

Proton nuclear magnetic resonance (¹ H NMR) spectra were determinedusing a Bruker AMX-400 spectrometer at 400 MHz. Chemical shifts arereported in parts per million (ppm) downfield from tetramethylsilane(TMS; δ=0 ppm) using residual CHCl₃ as a lock reference (δ=7.25 ppm).Multiplicities are abbreviated in the usual fashion: s=singlet;d=doublet; t=triplet; q=quartet; m=multiplet; br=broad.

Carbon nuclear magnetic resonance (¹³ C NMR) spectra were performed on aBruker AMX-400 spectrometer at 100 MHz with composite pulse decoupling.Samples were prepared as with ¹ H NMR spectra, and chemical shifts arereported relative to TMS (0 ppm); residual CHCl₃ was used as an internalreference (δ=77.0 ppm).

All high resolution mass spectral (HRMS) analyses were determined byelectron impact ionization (EI) on a JEOL JMS-DX 303HF mass spectrometerwith perfluorokerosene (PFK) as an internal standard. Low resolutionmass spectra (MS) were determined by either electron impact ionization(EI) or chemical ionization (CI) using the indicated carrier gas(ammonia or methane) on a Delsi-Nermag R-10-10 mass spectrometer. Forgas chromatography/mass spectra (GCMS), a DB-5 fused capillary column(30 m, 0.25 mm thickness) was used with helium as the carrier gas.Typical conditions used a temperature program from 60-250° C. at 40°C./min.

Thin layer chromatography (TLC) was performed using precoated glassplates (silica gel 60, 0.25 mm thickness). Visualization was done byillumination with a 254 nm UV lamp, or by immersion in anisaldehydestain (9.2 mL p-anisaldehyde in 3.5 mL acetic acid, 12.5 mL conc.sulfuric acid and 338 mL 95% ethanol (EtOH)) and heating tocolorization.

Flash silica gel chromatography was carried out according to thestandard protocol.

Unless otherwise noted, all solvents and reagents were commercial gradeand were used as received, except as indicated hereinbelow, wheresolvents were distilled under argon using the drying methods listed inparantheses: CH₂ Cl₂ (CaH₂); benzene (CaH₂); THF (Na/ketyl); Et₂ O(Na/ketyl); diisopropylamine (CaH₂).

    ______________________________________                                        Abbreviations                                                                 ______________________________________                                        OTf         triflate                                                          TLC         thin layer chromatography                                         EtOAc       ethyl acetate                                                     TIPS        triisopropylsilyl                                                 PMB         p-methoxybenzyl                                                   Bn          benzyl                                                            Ac          acetate                                                           hex         hexane                                                            THF         tetrahydrofuran                                                   coll        collidine                                                         LiHMDS      lithium hexamethyldisilazide                                      DMF         N,N-dimethylformamide                                             DMAP        2-dimethylaminopyridine                                           DDQ         2,3-dichloro-5,6-dicyano-1,4-benzoquinone                         TBAF        tetra-n-butylammonium fluoride                                    M.S.        molecular sieves                                                  r.t.        room temperature                                                  r.b.        round bottom flask                                                ______________________________________                                    

EXAMPLE 1

Preparation of Polymer-Bound Glucal 18:

Polymer-bound galactal 7 (500 mg; S. J. Danishefsky, et al., J. Am.Chem. Soc. 1992, 8331) was placed in a 100 mL polymer flask and dried invacuo. On cooling to 0° C. under N₂, dry CH₂ Cl₂ (20 mL) and freshlyprepared Murray solution (30 mL; R. W. Murray and R. Jeyaraman, J. OrgChem. 1985, 2847) was added. After stirring at 0° C. for ˜90 min.,solubles were filtered using N₂ pressure. The oxidation procedure wasrepeated. The resulting epoxide of 7 kept on a vacuum line for ˜3 h todry. A solution of glucal 19 (1.0 g in 8 mL dry THF) was added, and themixture was cooled to -23° C. (dry ice-CCl₄). A solution of ZnCl₂ in THF(0.8 mL 1.0 M) was added. The mixture was slowly allowed to warm to r.t.(over ˜2 h), and then stirred at r.t. overnight. The polymer-boundglucal 18 was rinsed with 3×20 mL THF, and dried on a vacuum line.

Preparation of Polymer-Bound Tetrasaccharide 20:

Polymer-bound glucal 18 and Sn(OTf)₂ (0.80 g, 1.92 mmol) were combinedand dried in vacuo. On cooling to 0° C. under N₂, a solution of fucosyldonor 10 (1.8 g, 4.1 mmol) in 20 mL dry THF with di-t-butylpyridine (1.7mL, 7.57 mmol) was added. The mixture was allowed to warm slowly tor.t., and stirred overnight. The polymer was washed with 2×20 mL dryTHF, 2×20 mL dry dioxane, 20 mL DMSO, and 2×20 mL THF. The resultingpolymer-bound tetrasaccharide 20 was kept on a vacuum line to dry.

Preparation of Tetrasaccharide Glycal 21:

The polymer-bound tetrasaccharide 20 (50 mg) was stirred in 2 mL THF,and treated with 0.2 mL each of 1.0 M solutions of TBAF and AcOH in THF.The mixture was stirred at 40° C. overnight. The polymer was washed with3×5 mL THF. The combined rinsings were concentrated andcolumn-chromatographed on silica (2:1 EtOAc:hex), providingtetrasaccharide glycal 21 as a colorless gum. Yield: 9.0 mg.

EXAMPLE 2

Preparation of Diol 18':

Galactal 7' (0.100 g, 0.304 mmol) in 5 mL dry CH₂ Cl₂ at 0° C. under aN₂ atmosphere was treated with 10 mL Murray solution (freshly prepared)and stirred at 0° C. for 40 min. TLC (1:1 EtOAc:hex) showed no trace of7'. Solvents were evaporated using a dry N₂ stream. The residual epoxideof 7' was kept on a vac. line ˜2 h. To the epoxide under a N₂ atmospherewas added a solution of glucal derivative 3' (0.150 g, 0.496 mmol) in 3mL dry THF. On cooling to -78° C., 1.0 M ZnCl₂ in Et₂ O (0.50 mL, 0.50mmol) was added. The mixture was allowed to slowly warm to r.t. (over ˜2h) and stirred overnight. TLC (1:1 EtOAc:hex) showed that the reactionwas complete. Saturated aq. NaHCO₃ (20 mL) was added, and the mixturewas then extracted with EtOAc (3×20 mL). The organic layer was driedover MgSO₄. Column chromatography on silica (1:3 EtOAc:hex) affordeddiol 18' as a colorless solid. Yield: 173 mg (89%). [α]_(D) ²³ -9.8° (c1.0, CH₂ Cl₂).

Preparation of Tetrasaccharide 22:

Diol 18' (86 mg, 0.133 mmol) and fucosyl donor 10 (0.290 g, 0.665 mmol)were azeotropically dried using benzene. The mixture was dissolved in 3mL dry THF together with 0.65 mL di-t-butylpyridine and then added viacanula to a flask containing Sn(OTf)₂ (0.30 g, 0.72 mmol) and 4 Å MS(500 mg) at 0° C. under N₂ atm. The mixture was stirred at 0° C. ˜7 h.TLC (1:3 EtOAc:hex) shows no trace of diol 18'. The mixture waspartitioned between saturated aq. NaHCO₃ (100 mL) and EtOAc (2×100 mL).The organic layer was dried over MgSO₄. The organic layer was filteredthrough silica using EtOAc to obtain crude material, which was thenpurified by chromatography on silica (1:9 EtOAc:hex) affordingtetrasaccharide 22. Yield: 170 mg (86%).

Preparation of Iodosulfonamide 23:

Procedure 1.

Tetrasaccharide glycal 22 (120 mg, 81.1 mmol) and PhSO₂ NH₂ (20 mg, 0.13mmol) were azeotropically dried using benzene. Added (glove bag) 4 Å MS(0.2 g). After cooling to 0° C. under N₂, dry CH₂ Cl₂ (1.0 mL) wasadded. The mixture was treated with a solution of I(coll)₂ ClO₄(prepared from 100 mg Ag(coll)₂ ClO₄, 5 mL collidine, and 60 mg I₂ in 1mL dry CH₂ Cl₂) via canula through a plug of flame-dried celite and 4 ÅMS. The mixture was stirred at 0° C. for 40 min. TLC (1:4 EtOAc:hex)showed iodosulfonamide 23 as the major component. The mixture wasfiltered through celite, which was rinsed with Et₂ O. The organic layerwas extracted with saturated aq. Na₂ S₂ O₃, saturated aq. CuSO₄, brine,and then dried over MgSo₄. Column chromatography on silica (1:4EtOAc:hex) gave iodosulfonamide 23 as a colorless solid. Yield: 115 mg(80%).

Procedure 2.

Tetrasaccharide glycal 22 (200 mg, 0.135 mmol), PhSO₂ NH₂ (42 mg, 0.27mmol), and 200 mg powdered 4 Å MS in 2.0 mL dry CH₂ Cl₂ at 0° C. under aN₂ atmosphere was treated with I(coll)₂ ClO₄ (prepared from 120 mgAg(coll)₂ ClO₄ and 67 mg I₂ in 1 mL dry CH₂ Cl₂). The mixture wasstirred at 0° C. (protected from light using foil) for 30 min. TLC (1:2EtOAc:hex) showed mainly iodosulfonamide with some glycal.

After ˜1 h more at 0° C., TLC showed no noticeable improvement. Themixture was filtered through celite, which was washed with Et₂ O. Afterextracting with saturated aq. Na₂ S₂ O₃, saturated aq. CuSO₄, brine, theorganics were dried over MgSO₄. Column chromatography on silica (1:3EtOAc:hex) gave 23 as a colorless solid. Yield: 165 mg (69%). [α]_(D) ²³=-85.7° (c 1.0, CH₂ Cl₂).

Preparation of Hexasaccharide 25:

Iodosulfonamide 23 (60 mg, 34 mmol) in a 35 mL r.b. was treated with 200mg powdered 4 Å MS (glove bag). To this flask under N₂ was added asolution of protected lactal 24 in THF (1.5 mL). On cooling the mixtureto -78° C., a solution of AgBF₄ (40 mg, 0.206 mmol) was added in 0.25 mLdry THF. The mixture was stirred and slowly warmed to r.t. overnight.The mixture was warmed to 45° C. and stirred ˜36 h. TLC showed only atrace of iodosulfonamide. Saturated aq. NH₄ Cl (5 mL) was added, and themixture was extracted with 3×10 mL EtOAc. The organic layer was driedover MgSO₄. Column chromatography on silica (1:3 EtOAc:hex) afforded 25as a colorless oil. Yield: 42 mg (55%). [α]_(D) ²³ =-33.8° (c 2.0, CH₂Cl₂)

Preparation of Hexasaccharide 25a:

Hexasaccharide 25 (55 mg, 24.4 mmol) in ˜1.5 mL THF was treated at 0° C.with TBAF (0.25 mL, 1.0 M solution in THF, 0.25 mmol), and stirred atr.t. overnight. TLC (1:9 MeOH:CH₂ Cl₂) showed a 3:1 mixture of 25a vs. aless polar substance. Additional 1.0 M TBAF (0.10 mL) was added, and themixture was stirred overnight at r.t. TLC showed that the reaction wascomplete. Solvents were removed using a N₂ stream. Column chromatographyon silica (1:19 MeOH:CH₂ Cl₂) afforded a ˜1:2 mixture corresponding totwo compounds which differ only in the presence or absence of a3,4-cyclic carbonate group. Crude yield: 35 mg total weight for twoproducts. The crude mixture was used as such for the next reaction.

Preparation of Peracetylated Hexasaccharide 26:

Hexasaccharide 25a (36 mg) in 0.25 mL dry THF was added via canula to ˜8mL bright blue Na/NH₃ solution at -78° C (dry ice bath) under N₂ atm.After removing the dry ice bath, the mixture was stirred in refluxingNH₃ (dry ice condenser) for 15 min. After adding 2 mL dry MeOH(slowly!), the resulting mixture was stirred while blowing off NH₃ witha M₂ stream. The MeOH solution was treated with Dowex 50×8 [H⁺ ] untilpH ˜8-9, and then filtered. The resin was washed with MeOH. The residuewas concentrated and kept on a vacuum line to dry. Under a N₂atmosphere, the residue was treated with 1 mL dry pyridine and 0.5 mLAc₂ O, and stirred at r.t. overnight. TLC (EtOAc) showed thathexasaccharide 26 is major component. Upon concentration, the residuewas purified by column chromatography on silica (1:4 hex:EtOAc).

Preparation of Hexasaccharide 17:

Hexasaccharide 26 (10.0 mg, 6.3 mmol) under N₂ at 0° C. was treated with0.5 mL dry CH₂ Cl₂. 3,3-dimethyldioxirane solution (0.20 mL) was added,and the mixture was stirred at 0° C. ˜40 min. TLC (EtOAc) showed notrace of 26. Solvents were evaporated with a N₂ stream. The epoxide wasdried on a vacuum line for ˜2 h. The epoxide was treated under a N₂atmosphere with 0.5 mL allyl alcohol (passed through basic alumina todry) and 0.5 mL dry THF. On cooling to -78° C., 1.0 M ZnCl₂ (10 mL) indry Et₂ O was added. After warming slowly to r.t., the mixture wasstirred overnight. Saturated aq. NaHCO₃ (5 mL) was added, and themixture was extracted with 3×5 mL EtOAc. The combined organic layerswere dried over MgSO₄, filtered and concentrated to an oil, which wasdried on a vacuum line for ˜2 h. The residue was treated to pyridine:Ac₂O (2:1, 1.5 mL) while stirring overnight. Solvents were removed, and theresidue was purifed by column chromatography on silica (1:4 hex:EtOAc),affording hexasaccharide 17 as a colorless solid. Yield: 5.5 mg.

Results and Discussion

A Highly Convergent Synthesis of the Lewis Y Blood Group Determinant inConjugatable Form

Construction of the Le^(y) determinant commences with lactal (1a) (W. N.Haworth, E. L. Hirst, M. M. T. Plant, R. J. W. Reynolds, J. Chem. Soc.1930, 2644) as shown in FIG. 2. Capping both primary hydroxyl groups astheir TBDPS ethers under standard conditions was followed by simpleengagement of the 3' and 4' hydroxyl functions as a cyclic carbonate 2a.The stereospecific introduction of two α-linked fucose residues gavetetrasaccharide glycal 3a in 51% yield in a single step. The donor usedwas the known fluorosugar 5a (S. J. Danishefsky, J. Gervay, J. M.Peterson, F. E. McDonald, K. Koseki, T. Oriyama, D. A. Griffith, C-H.Wong, D. P. Dumas, J. Am. Chem. Soc. 1992, 114, 8329) following amodification of the original Mukaiyama conditions. (T. Mukaiyama, Y.Murai, S. Shoda, Chem. Lett. 1981, 431) Glycal 3a corresponds to theLe^(y) hapten, lacking the N-acetyl function in the glucose residue. Theproblem was then to introduce this group as well as a galactose spacermodule.

Methodology developed previously (D. A. Griffith, S. J. Danishefsky, "Onthe Sulfonamidoglycosylation of Glycals. A Route to OligosaccharidesWith 2-Aminohexose Subunits+", J. Am. Chem. Soc. 1990 112, 5811) provedappropriate to attain these goals. Glycal 3a was treated with iodoniumdicollidine perchlorate and benzenesulfonamide to afford iodosulfonamide4a. Azaglycosylation using the 3-stannyl ether of galactal (9a) (S. J.Danishefsky, K. Koseki, D. A. Griffith, J. Gervay, J. M. Peterson, F. E.McDonald, T. Oriyama, J. Am. Chem. Soc. 1992, 114, 8331) in the presenceof silver tetrafluoroborate gave pentasaccharide glycal Ga in 75% yieldas shown in FIG. 3. Having 6a in hand, one can iterate theazaglycosylation sequence or activate the glycal as its epoxide andcontinue with further glycosylations. To demonstrate the ability tofashion a conjugatable form of Le^(y) hapten, formation of the allylglycoside was important. The feasibility of converting the sulfonamidogroup into the target acetamide was demonstrated. Glycal 6a wasdeprotected in two steps as shown. Peracetylation afforded acetamidoglycal 7a. Activation of the glycal as its epoxide with3,3-dimethyldioxirane (R .L. Halcomb, S. J. Danishefsky, J. Am. Chem.Soc. 1989, 111, 6661), followed by epoxide opening with allyl alcohol inthe presence of zinc chloride gave the desired peracetylated β-allylpentasaccharide which was deacetylated by action of methoxide to providethe target Le^(y) hapten as its β-allyl glycoside 8a. (8a [α]_(D) -72.70(c. 1 MeOH); IR (thin film) 3350, 2940, 2900, 2830, 1650, 1550, 1365,1300, 1155, 1070, 1030; ¹ H NMR (400 MHz, CD₃ OD) δ 5.95 (m, 1H), 5.32(d, J=17.25 Hz, 1H), 5.14-5.19 (m, 2H), 5.04 (d, J=3.83 Hz, 1H), 5.02(d, J=3.50 Hz, 1H). 4.68 (d, J=8.15 Hz, 2H), 4.51 (d, J=5.70 Hz, 1H)3.40-4.38 (m, 27H). 1.96 (s, 3H), 1.23 (m, 6H); HRMS (FAB) cald for C₃₅H₅₆ NO₂₄ Na 900.3325 found 900.3310) The aldehyde, derived by ozonolysisof 8a, could be conjugated to a carrier protein by the method ofBernstein and Hall.

This synthesis is the most direct route to the Le^(y) determinant known.(O. Hindsgaul, T. Norberg, J. Le Pendu, R. U. Lemieux, Carbohydr Res.1982, 109, 109; U. Spohr, R. U. Lemieux ibid, 1988, 174, 211; forprevious syntheses, see: J. C. Jacquinet, P. Sinay, J. Org. Chem. 1977,42, 720; S. Nilsson, H. Lohn, T. Norberg, Glycoconjugate J. 1989, 6, 21;R. R. Schmidt, A. Topfer, Tetrahedron Lett. 1991, 32, 3353; W. Kinzy, A.Low, Carbohydrate. Res. 1993, 245, 193) The method is stereospecific ateach step, and it illustrates the versatility of glycals both as donorsand acceptors and takes advantage of 1,2-glycal epoxides and theirpresumed N-sulfonylaziridine counterparts. The method also makespossible extensive analog preparation and variation of conjugationstrategies.

The synthesis of 3a and 6a are shown below:

3a: To 2.00 g (2.47 mmol) of lactal carbonate 2a was added 4.44 g (9.86mmol) of fucosyl fluoride 5a. The mixture was azeotroped 5 times withbenzene and placed under high vacuum for two hours. Under an argonatmosphere 2.77 ml (12.33 mmol) of di-tert-butyl pyridine and 16 ml ofdry ether were added. 2.0 g of freshly activated 4A molecular sieveswere added and the mixture stirred one hour at room temperature. In anargon glove bag, 2.34 g (12.33 mmol) of stannous chloride (SnCl₂) and2.56 g (12.33 mmol) of silver perchlorate (AgClO₄) were added. The flaskwas equipped with a reflux condensor and the reaction brought to refluxfor 72 hours. The reaction was quenched with 5 ml of saturatedbicarbonate and filtered through a pad of celite. Diluted with 50 mlethyl acetate and washed 2 times with sat. bicarbonate, 2 times withsat. copper sulfate and 2 times with sat. brine. The organics were driedover MgSO₄ and concentrated. Flash chromatography in 20% ethylacetate/hexanes afforded 2.10 g (51%) of a white foam 3a: [α]_(D) -78.⁹(c.555,CHCl₃); IR (thin film) 3040, 3000, 2905, 2860, 2830, 1820, 1800,1710, 1635, 1585, 1570, 1480, 1460, 1440, 1415, 1370, 1350, 1300, 1260,1205, 1145, 1100, 950, 735, 695, ¹ H NMR (400 MHz, CDCl₃) δ 8.09 (d,J=8.12 Hz, 2H), 8.00 (d, J=8.26 Hz, 2H) 7.66 (m, 4H), 7.59 (d=J=6.74Hz,4H), 7.56 (t, J=7.27 Hz, 1H), 7.30-7.50 (m,22H) 7.16-7.26 (m,10H)7.09 (m,2H), 6.99 (t, J=7.59 Hz, 2H) 6.89 (t, J=7.97 Hz, 1H), 6.43 (d,J=6.08Hz, 1H), 5.46 (bs, 1H), 5.38 (bs, iH), 5.35 (d, J=3.42 Hz, 1H),4.89 (d, J=11.35 Hz, 1H), 4.75-4.80 (m, 4H), 4.72 (d, J=5.88 Hz, 2H),4.69 (d, J=4.27 Hz, 2H), 4.36-4.55 (m, 5H), 4.28 (q, J=6.51 Hz, 1H),4.17 (bd, J=5.46 Hz, 1H), 3.90-4.00 (m,6H), 3.85 (d, J=2.99 Hz, 1H),3.82 (d, J=2.89 Hz, 1H), 3.56-3.78 (m, 4H), 1.07 (m, 24H); HRMS (FAB) :calcd for C₉₉ H₁₀₆ O₂₀ Si₂ Na 1694.6740 found 1694.6787.

6a: 230 mg (0.12 mmol) of iodosulfonamide 4a was azeotroped 5 times withdry benzene and placed under high vacuum for two hours. Added 2.4 ml ofTHF solution of 15 eq. of tin ether 9a (generated by azeotrophic removalof water overnight with a Dean-Stark trap equipped with freshlyactivated 4A mol. sieves from 561 mg (1.80 mmol) of 6a-TIPS-galactal and673 μl (1.32 mmol) bis(tributylin) oxide in 80 ml of benzene). To thissolution stirring under an argon atmosphere was added 200 mg of freshlyactivated 4A powdered molecular sieves. Stirred one hour at roomtemperature. Cooled solution to -78° C. and added, via cannula, asolution of 187 mg (0.96 mmol) of silver tetrafluroborate in 2.4 ml ofTHF. Warmed to room temperature over 15 hours and quenched the reaction,which had turned bright yellow, with 2 ml. of sat. bicarbonate. Thereaction mixture was filtered through a pad of celite into a separatoryfunnel. The celite pad was washed thoroughly with ethyl acetate. Theorganics were washed twice with sat. bicarbonate and twice with sat.brine. The organics were dried over MgSO₄. Concentration andchromatography in 25% ethyl acetate/hexanes gave 193 mg (75%) as a whitefoam 6a: [α]_(D) -126.4° (c,505,CHCl₃), IR (thin film) 3500, 3040, 3000,2905, 2840, 1820, 1800, 1705,1635, 1590, 1440, 1410, 1255, 1195, 1100,1080, 1035, 815, 730, 695; ¹ H NMR (400 MHz, CDCl₃) δ 8.09 (app t, 4H),7.08-7.65 (m, 46H), 6.90 (t, J=7.65 Hz, 3H), 6.76 (d, J=6.91 Hz, 2H),6.12 (d, J=6.59 Hz, 1H), 5.50 (bs 1H), 5.45 (bs 1H), 5.28 (app t, 2H),3.03-4.91 (m, 36H), 1.09 (m, 45H); LRMS (FAB): cald for C₁₂₀ H₁₄₁ NO₂₆SSi₃ Na 2153 found 2153.

A Strategy for the Assembly of Complex, Branched Oligosaccharide Domainson a Solid Support: An Application to a Concise Synthesis of theLewis^(b) Domain in Bioconjugatable Form.

The assembly of the Le^(b) (type 1) domain is a relatively moredifficult undertaking than was the Le^(y) (type 2) target, whereinlactal was used as a convenient starting material. In the case of thetype 1 determinant, lactal is not a useful starting material. Thesynthesis of the Le^(b) system offered an opportunity to apply thepolymer-based oligosaccharide construction method. (S. J. Danishefsky,K. F. McCLure, J. T. Randolph, R. B. Ruggeri, Science 1993, 260, 1307)The strategy is summarized in FIG. 4, wherein polymer-bound glycal 1 isactivated for glycosyl donation via direct formation of a 1,2-anhydroderivative 2. Reaction of 2 with acceptor glycal 3 furnishes 4.Reiteration is achieved by means of direct epoxidation and reaction withacceptor 3. The self-policing nature of the method and the simple "onetime" purification at the end of the synthesis are useful features.

The present invention discloses an important additional dimension of thepolymer-bound method. The logic is captured by inspection of FIG. 5.Each glycosylation event generates a unique C₂ hydroxyl. In principle(and in fact, see infra) this hydroxyl can function as a glycosylacceptor upon reaction with a solution based donor. The glycal linkageof 5, still housed on the support, can be further elongated. In thisway, branching at C₂ is accomplished while minimizing the requirementfor protecting group machinations. (For an application of this strategyin the synthesis of a complex saponin, see: J. T. Randolph, S. J.Danishefsky, J. Am Chem Soc. 1993, 115, 8473)

In principle, this branching can be implemented at any site in a growingchain. For such an extension, it would be necessary to cap allpreviously generated hydroxyl groups generated on the "polymer side"(non-reducing end) of the growing domain. Thus, the polymer-boundligosaccharide can serve as either donor or acceptor, hereverappropriate.

Initial efforts at reduction to practice identified tetrasaccharideglycal 6, bearing H-type 2 blood group specificity, as a goal.Polymer-supported galactal 7 (using as polymer support polystyrenecrosslinked with 1% divinylbenzene functionalized using publishedprocedures: T-H. Chan, W.-Q. Huang, J. Chem. Soc., Chem. Commun. 1985,909; M. J. Farrall. J. M. J. Frechet, J. Org. Chem1976, 41, 3877)reacted with a solution of 3,3-dimethyldioxirane (R. W. Murray, R.Jeyaraman, J. Org. Chem. 1985, 50, 2847), to provide the corresponding1,2-anhydrosugar glycosyl donor, which was treated with a solution ofglucal derivative 8 in the presence of ZnCl₂ to provide 9 (R. L.Halcomb, S. J. Danishefsky, J. Am. Chem Soc. 1989, 111, 6661) Thispolymer-bound disaccharide acted as a glycosyl acceptor upon treatmentwith a solution of fucosyl fluoride 10 (K. C. Nicoloau, C. W. Hummel, Y.Iwabuchi, J. Am. Chem. Soc. 1992, 114, 3126) in the presence of Sn(OTf)₂thereby giving 11. Retrieval of the trisaccharide glycal from thesupport was accomplished using tetrabutylammonium fluoride (TBAF) toafford 12 in 50% overall yield from 7.

The trisaccharide, retrieved from the polymer, could then be furtherelaborated. Toward this end, compound 12 was converted to silyl ether 13by reaction with TIPSCl. The latter was converted to the iodosulfonamidederivative 14 by the action of I(coll)₂ ClO₄ in the presence of PhSO₂NH₂. Reaction of 14 with galactal stannyl ether derivative 15 in thepresence of AgBF₄ gave 16 77% yield. (D. A. Griffith, S. J. Danishefsky,J. Am. Chem Soc. 1990, 112, 5811) Tetrasaccharide glycal 16 wasdeprotected and peracetylated to afford 6. (S. J. Danishefsky, K.Koseki, D. A. Griffith, J. Gervay, J. M. Peterson, F. E. McDonald, T.Oriyama, J. Am. Chem Soc. 1992, 114, 8331)

Thus, the synthesis of the full H-type determinant was achieved bysequential polymer- and solution-based maneuvers. The next target wasthe more complex Le^(b) hexasaccharide 17. The campaign proceeded asshown in FIGS. 7a and 7b. Polymer-bound galactal 7 was converted to 18upon epoxidation with 3,3-dimethyldioxirane followed by reaction withglucal derivative 19. This disaccharide diol was then bisfucosylatedusing fucosyl donor 10 in the presence of Sn(OTf)₂ to afford 20.Retrieval from the support with TBAF provided 21, which was obtained in40% overall yield from 7. Compound 21 reacted with TIPSCl to give 22.

Iodosulfonamide 23, obtained from 22 using I(coll)₂ ClO₄ and PhSO₂ NH₂,reacted with lactal derivative 24 in the presence of AgBF₄ to providehexasaccharide glycal 25 in 55% yield. Deprotection of 25 wasaccomplished in two stages (TBAF to remove the silyl ethers, followed byNa/NH₃ reduction to remove the aromatic protecting groups), and thecrude product was peracetylated to give 26 in a 51% overall yield.Compound 26 was converted, via the 1,2-anhydrosugar derivative, to allylglycoside 17, which can be activated by ozonolysis to the aldehyde(R═CH₂ CHO) for subsequent coupling to a protein by the method ofBernstein and Hall.

In sum, the present invention extends the solid-support glycal assemblymethod for complex carbohydrate domain synthesis to include thebranching patterns critical for biorecognition. Specifically, thedeterminant for the binding of H. pylori to human gastric epithelium hasbeen stereospecifically fashioned, with simplicity, in a way whichprovides significant relief from some of the complexities of protectinggroup manipulations.

Experimental Procedure:

6: ¹ H NMR (400 MHz, CDCl₃); δ 6.39 (d, 1H, J=6.2 Hz, H₁ galactal), 5.65(d, 1H, J=8.9 Hz, NHAC), 5.35 (d, 1H, J=3.8 Hz), 5.33 (m, 1H), 5.29 (d,1H, J=2.6 Hz), 5.27 (d, 1H, J=3.1 Hz), 5.17-5.09 (m, 2H),4.97-4.90(m,2H), 4.81 (dd, 1H, J=3 Hz, J=6.1 Hz, H₂ galactal), 4.75 (d,1H, J=8.0 Hz), 4.52 (m, 1H), 4.48 (dd, 1H, J=12.0 Hz), 4.44-4.06 (m,8H), 3.88-3.77 (m, 4H). 3.61 (m, 1H), 2.18-1.97 (m, 33 H, COCH₃), 1.18(d, 3H, J=6.5 Hz, CH₃ fucose); ¹³ C NMR (CDCl₃): δ 170.80, 170.77,170.72, 170.67, 170.62, 170.34, 170.21, 170.09, 170.01, 169.99, 169.65,144.92 (C₁ galactal), 100.22, 98.83, 98.58, 95.55, 74.48, 73.38, 73.13,73.06, 71.48, 71.01, 70.68, 67.97, 67.42, 67.18, 67.05, 65.94, 64.83,62.35, 62.22, 60.88, 60.37, 54.21, 23.23, 22.15, 20.85, 20.82, 20.79,20.76, 20.65, 20.61, 20.57, 15.51, (C₆ fucose); IR (thin film): 3368.7(NH), 2965.6, 2934.6, 1746.5 (C═O), 1537.5, 1435.9, 1371.3, 1228.5,1065.0, 1046.0; [α]_(D) ²³ =-51.1° (c 1.8, CH₂ Cl₂); HRMS (FAB); calcd.for C₄₆ H₆₃ NNaO₂₈ : m/z=1100.3434, found 1100.3436.

21: Polymer-bound galactal 7 (loading=0.85 mmol glycal/g), which hadbeen placed in a round-bottom flask equipped with a fritted outlet, wassuspended in CH₂ Cl₂ under N₂, cooled to 0° C., and then treated with asolution of 3,3-dimethyldioxirane. The mixture was stirred(teflon-coated magnetic stir bar) for 40 min. at 0° C., after which timesolubles were removed by filtration through the fritted outlet (N₂pressure). The polymer bound 1,2 anhydrosugar was evacuated (ca. 0.1torr) for several hours in order to dry the material for the next step.This material was once again placed under N₂ before being treated with19 (˜10 molar equivalents as a 0.5 M solution in THF). The suspensionwas cooled to -40° C., and treated with ZnCl₂ (˜2 molar equivalents as a1.0 M solution in THF). The reaction mixture was allowed to slowly warmto rt (over ca. 2 h), and then stirred an additional 3-4 h. Solubleswere removed by filtration, and polymer 18 was washed several times withTHF and then dried in vacuo. To compound 18 was added, in a glove bag,solid Sn(OTf)₂ (˜ molar equivalents), and the mixture was placed underN₂ and cooled to 0° C. before being treated with 10 (˜5 molarequivalents as a 0.2 M solution in THF and di-tert-butylpyridine (˜8molar equivalents). The suspension was allowed to warm to rt and stirred8-10 h. The mixture was rinsed with anhydrous THF (2 times), 1,4-dioxane(2 times), again with THF, and then dried in vacuo. Compound 20 (100 mg)was suspended in THF, treated with a 1:3 mixture of AcOH and TBAF (˜0.2M in TBAF, ˜10 molar equivalents), and the mixture was stirred for 18 hat 40° C. The polymer was rinsed with THF (3 times), and the combinedrinsings were concentrated and purified by column chromatography onsilica gel (1:1 EtOAc: hexanes). Compound 21 (18 mg) was obtained as acolorless solid (40% overall yield from 7): ¹ H NMR (400 MHz, CDCl₃): δ7.40-7.25 (m, 30H, Ar H), 6.18 (d, 1H, J=6.0 Hz, H₁ glucal), 5.26 (d,1H, J=3.5 Hz, H₁ fucose), 5.09 (d, 1H, J=3.7 Hz, H₁ fucose), 4.96 (t,2H, J=10.8 Hz, PhCH₂), (4.90-4.56 (m, 13H), 4.43 (m, 1H), 4.15-4.06 (m,4H), 3.97 (dt, 1H, J=8.3 Hz, J=2.4 Hz), 3.87-3.65 (m, 10H), 3.64 (d,1H), 3.57 (d, 1H), 2.69 (br, 1H, OH), 2.52 (br, 1H, OH), 1.11 (d, 3H,J=7.0 Hz, CH₃ fucose), 1.09 (d, 3H, J=7.0 Hz, CH₃ fucose); ¹³ C NMR(CDCl₃); δ 153.37 (C═O), 145.75 (C₁ glucal), 138.60, 138.52, 138.19,137.61, 128.55, 128.52, 128.44, 128.24, 128.16, 128.07, 127.62, 127.56,127.45, 98.71, 98.38, 97.65, 97.34, 79.26, 78.87, 78.67, 78.01, 77.79,77.65, 76.37, 76.10, 74.92, 74.40, 74.16, 73.95, 72.86, 72.64, 72.53,67.43, 67.29, 61.31, 60.90, 16.65 (C₆ fucose), 16.53 (C₆ fucose); IR(thin film): 3467.0 (OH), 3029.6, 2923.6, 1807.2 (C═O), 1647.3, 1496.0,1453.5, 1358.1, 1240.2, 1095.6, 1049.2, 738.5, 697.2; [α]_(D23) =-82.5°(c 0.4, CH₂ Cl₂); HRMS (FAB); calcd. for C₆₇ H₇₄ NaO₁₈ : m/z=1189.4772,found 1189.4757.

25: To a mixture of 23 (60 mg, 34 μmol) and powdered 4A molecular sieves(200 mg) under N₂ was added, via canula, a solution of 24 (0.21 mmol) inanhydrous THF (1.5 mL). The stirred suspension was cooled to -78° C.before being treated with a solution of AgBF₄ (0.21 mmol) in 0.25 mL ofanhydrous THF. The mixture was stirred and allowed to slowly warm to rtovernight. The suspension, which had developed a bright-yellow color,was heated, with stirring, at 45° C. for an additional 36 h, until theTLC (2.5 EtOAc:hexanes) showed no trace of 23. The mixture was treatedwith saturated aqueous NH₄ Cl (5 mL) and then extracted with EtOAc (3×10mL), and the organics were dried over MgSO₄. The crude product waspurified by silica gel chromatography (1:3 EtOAc:hexanes) to give 25 asa colorless oil (42 mg, 55%): ¹ H NMR (400 MHz, acetone-d₆): δ 8.17(d,2H, J=7.3 Hz, PhSO₂), 7.50-7.20 (m, 33H, ArH), 6.52 (d, 1H, J=10.5 Hz,NH), 6.30 (dd, 1H, J=6.0 Hz, H₁ glucal), 5.35-5.32 (m, 2H), 5.25 (d, 1H,J=7.9 Hz), 5.15 (m, 2H), 4.99-4.92 (m, 3H), 4.86-4.52 (m, 14H), 4.45(dd, 1H, J=7.91 Hz, J=2.4 Hz), 4.32-4.23 (m, 3H), 4.22 (dd, 1H), 4.17(d, 1H, J=10.1 Hz), 4.08-3.84 (m, 18H), 3.79-3.73 (m, 2H), 3.66 (m, 1H),3.55 (t, 1H, J=6 Hz), 3.50 (dd, 1H, J=9.7 Hz), 1.33 (d, 3H, J=6.5 Hz,CH₃ fucose), 1.31 (d, 3H, J=6.4 Hz, CH₃ fucose), 1.20-0.98 (m, 84H,3×Si(i-Pr)₃); ¹³ C NMR (acetone-d₆): 145.66 (C═O), 132.72, 131.48,131.45, 131.28, 131.16, 130.77, 130.48, 121.31, 120.11, 119.86, 119.78,119.25, 95.63, 94.70, 91.37, 89.64, 89.31, 86.52, 73.38, 72.24, 71.00,70.71, 70.37, 69.80, 69.59, 69.06, 68.23, 67.92, 67.38, 67.10, 66.49,65.67, 65.33, 64.60, 64.34, 64.03, 63.45, 63.30, 59.46, 58.83, 58.37,54.45, 53.32, 49.86, 19.67 (C₆ fucose), 18.42 (C₆ fucose), 9.55, 9.48,9.45, 9.31, 9.23, 3.82, 3.70, 3.64; IR (thin film): 3491.9 (OH), 3030.1,2941.2, 2865.5, 1835.8, 1819.5, 1649.8, 1496.2, 1462.3, 1349.9, 1245.5,1155.2, 1095.1, 1049.4, 882.2, 734.8, 692.0; [α]_(D23) =-33.8° (c 2.0,CH₂ Cl₂); HRMS (FAB): calcd for ¹² C₁₂₀ ¹³ CH₁₇₉ NNaO₂₉ SSi₄ :m/z=2278.1292, found 2278.1296.

17: ¹ H NMR (400 MHz, CD₃ OD): δ 6.00 (m, 1H, J=5.6 Hz, CH₂ CH═CH₂),5.37 (dd, 1H, J=1.6 Hz, J=7.3 Hz, CH₂ CH═CH₂), 5.20 (dd, 1H, J=1.6 Hz,J=9.5 Hz, CH₂ CH═CH₂), 5.18 (d, 1H, J=3.9 Hz, H₁ fucose), 5.10 (d, 1H,J=3.8 Hz, H₁ fucose), 4.64 (d, 1H, J=6.9 Hz), 4.45 (d, 1H, J=7.4 Hz),4.43-4.23 (m, 2H), 4.27 (dd, 1H, J=9.3 Hz, J=10.6 Hz), 4.23-4.11 (m,2H), 4.02-3.29 (m, 31H), 2.06 (s, 3H, NAc), 1.31 (d, 3H, J=6.6 Hz, CH₃fucose, 1.29 (d, 3H, J=6.6 Hz, CH₃ fucose); ¹³ C NMR (CD₃ OD): δ 173.20(C═O), 135.73 (CH₂ CH═CH₂), 105.13, 103.30, 102.49, 101.62, 99.63,96.86, 80.79, 80.67, 78.44, 76.67, 76.49, 75.89, 74.80, 74.59, 73.94,73.61, 73.40, 71.55, 71.38, 71.16, 70.42, 70.26, 70.14, 67.77, 67.30,67.21, 62.79, 62.34, 61.99, 55.54, 22.97 (NAc), 16.65 (2 C's, C₆fucose); IR (thin film): 3376.6 (OH), 2924.2, 1652.5 (C═O), 1383.1,1032.4; [α]_(D23) =-12.8° (c 0.25, MeOH); HRMS (FAB): calcd. for C₄₁ H₆₉NNaO₂₉ : m/z=1062.3853, found 1062.3837

Glycal Assembly Method Applied to the Synthesis of Human BreastTumor-Associated Antigen

The present invention provides a convergent synthesis of thehexasaccharide wherein the two trisaccharide domains have beenefficiently assembled in forms readily ammenable for coupling. Thesynthesis of the ABC trisaccharide is presented in FIG. 8. The α-linkageof this trisaccharide might be formed by employing a fluoro-sugar donor4b, using established conditions. (Gordon, D. M.; Danishefsky, S. J.,Carbohydr. Res., 1990, 206, 361-366.) Preparation of the appropriatedisaccharide acceptor commenced with 5b (Danishefsky, S. J.; Behar, V.;Randolph, J. T.; Lloyd, K. O., J. Am. Chem. Soc., 1995, 5701), itselfobtained from a glycal coupling.

Benzylation followed by desilylation, carbonate removal and selectivedibenzylation afforded the disaccharride 6b. The acceptor thus obtainedwas reacted with the fluorosugar 4b using modified Mukaiyama conditions(Mukaiyama, T.; Murai, Y.; Shoda, S., Chem. Lett., 1981, 431-433) toprovide the trisaccharide glycal 7b. Deprotection of the PMB etherprovided the ABC trisaccharide 8b, which was poised for coupling with asuitable DEF trisaccharide donor.

The synthesis of the DEF trisaccharide is described in FIG. 9.Epoxidation of the galactal 9b and standard coupling (Halcomb, R. L.;Danishefsky, S. J., J. Am. Chem. Soc., 1989, 111, 6661-6666.) withacceptor 10b afforded, regioselectively, the disaccharide 11b.Fucosylation employing the fluoro-fucose 12b (Dejter-Juszynski, M.;Flowers, H. M., Carbohydr. Res., 1973, 28, 61) provided a 5:1 ratio ofmonoglycosylation regioisomers, the major isomer being the desiredtrisaccharide 13b. This material was treated under standard conditionsto afford the trans-diaxial iodosulfonamide 14b.

Direct coupling reactions (Griffith, D. A.; Danshefsky, S. J., J. Am.Chem. Soc., 1990, 112, 5811-5819; Danishefsky, S. J.; Koseki, K.;Griffith, D. A.; Gervay, J.; Peterson, J. M.; McDonald, F. E.; Oriyama,T., J. Am. Chem. Soc., 1992, 114, 8331-8333) employing iodosulfonamidessuch as 14b with ABC trisaccharide acceptors failed, leading to adifferent donor functionality in the trisaccharide. In practice, theiodosulfonamide 14b was treated with excess lithium ethanethiolate toafford the ethyl thioglycoside 15b (FIG. 10). Precedent established bythe present inventors lead to the prediction of sulfonamideparticipation to provide the desired β-linked product from 15b.(Griffith, D. A., Ph.D. Thesis, Yale University, 1992) When donor 15bwas treated with MeOTf in the presence of acceptor 8b, a 10:1 mixture ofhexasaccharide isomers was obtained. The major product 16b was obtainedin 70-85% yield.

Ceramide attachment and elaboration commenced with epoxidation of 16b,followed by reaction with the stannyl ether 17b promoted by Zn(OTf)₂.(Liu, K. K.-C.; Danishefsky, S. J., J. Am. Chem. Soc., 1993, 115,4933-4934) Although the yield of this ceramide coupling is low, whenthis reaction was performed on trisaccharide 7b, the correspondingproduct was obtained in 66% yield. This material can then be used toobtain 18b. Following acetylation, the ceramide side-chain waselaborated by reduction of the azide functionality using Lindlar'scatalyst under an atmosphere of H₂ in the presence of palmitic anhydrideto provide 18b. Desilylation and saponification was followed bydissolving metal deprotection and MeOH quench. Peracetylation of thecrude mixture, followed by saponification provided the glycosphingolipid1b. Only the chemical shifts and coupling constants of the anomericprotons have been reported for the natural material. The spectrum ofsynthetic 1b is in complete agreement with this data. Furthermore, theproduct was characterized by exact mass, and ¹ H and ¹³ C NMR. Thesynthetic material has also been shown to bind to the monoclonalantibody MBr1.

In addition, the present invention provides the corresponding allylglycoside (FIG. 11). Deprotection of 16b, as above, and acetylationafforded the peracetate of the hexasaccharide glycal. Epoxidation,reaction with allyl alcohol, and saponification provided the allylglycoside 19b.

As in the case of the Le determinant, ozonolysis of the allyl group of19b will set the stage for reductive coupling to lysine residues ofproteins.

Synthesis of 3b:

3-0-(4-Methoxybenzyl)-D-galactal

A suspension of D-galactal (2b) (3.70 g, 25.3 mmol) and dibutyltin oxide(6.30 g, 1.0 equiv) in dry benzene (150 mL) was heated to reflux for 2 hwith azeotropic removal of water. The reaction was cooled and treatedwith PMBC1 (3.80 mL, 1.1 equiv) and tetrabutylammonium bromide (9.10 g,1.1 equiv) and refluxed for 4 h. The reaction was filtered throughsilica column and eluted with EtOAc/hexanes (4:1). Fractions containingproduct were concentrated and the residue triturated in hexanes to give4.50 g (67%) of product as white crystalline solid.

mp (hexanes) 117-118° C.; (a)²³ =-23.0° (CHCI₃, c=1.1); IR (KBr) 3313(br), 1645, 1513, 1228, 1082, 821 cm⁻¹ 1 H-NMR (400 MHz, CDCl₃) δ 7.28(2H, d, J=8.4 Hz), 6.89 (2H, d, J=8.4 Hz), 6.44 (1H, dd, J=6.4 Hz), 4.70(1H, dt, J=6.3, 1.9 Hz), 4.59-4.52 (2H, ABq, J=11.4 Hz), 4.20-4.18 (1H,m), 4.04-3.97 (1H, m), 3.90-3.82 (2H, m), 3.81 (3H, s), 2.73 (1H, d,J=3.1 Hz, C4--OH), 2.54 (1H, dd, J=8.2, 4.2 Hz, C6--OH); ¹³ C-NMR (100MHz, CDCl₃) δ 159.46, 145.02, 142.05, 129.46, 113.95, 99.36, 76.12,70.17, 70.14, 63.65, 62.74, 55.26; LRMS(NH₃) 284 (M+NH₄)⁺, 266 (M)⁺,249.

4,6-di-O-benzyl-3-0-(4-methoxybenzyl)-D-galactal (3b).

A solution of 3-0-(4-methoxybenzyl)-D-galactal (2.28 g, 8.56 mmol) andbenzyl bromide (3.75 mL, 3.68 mol equiv; freshly passed through basicalumina) in DMF (30 mL) under N² at 0° C. was treated with NaH (1.37 g,4.0 mol equiv) in two portions. The reaction was stirred 0.5 h at 0° C.and 1 h at rt. The reaction was carefully poured into 50 g of crushedice, diluted to 100 mL with water, then extracted with EtOAc-hexanes(1:1, 100 mL×3). Organic extracts were washed with water (100 mL×2),dried (Na₂ SO₄) and concentrated. Flash chromatography with 15% EtOAc-hexanes gave 3.58 g (94%) of the title compound as a clear liquid.

[α]²³ _(D) =-48.2° (CHCI3, c=0.85); IR (neat) 3030, 2867, 1645, 1613,1513 1247, 1092, 821, 736 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃) δ7.34-7.23(12H, m), 4.62 (1H, d, J=12.0 Hz), 4.59-4.51 (2H, ABq, J=11.7Hz), 4.50-4.39 (2H, ABq, J=11.9 Hz) ¹³ C-NMR (100 MHz, CDCl³) δ 159.04,143.99, 138.30, 137.90, 130.43, 128.26, 128.20, 128.03, 127.77, 127.57,127.56, 113.67, 100.00, 75.58, 73.28, 73.17, 71.13, 70.42, 70.28, 68.35,55.15; LRMS (NH₃) 464 (M+NH⁴⁺, 100), 326 (18), 309 (48), 253 (17).

Synthesis of 4b:

A solution of galactal 3b (3.20 g, 7.17 mmol) in dry CH₂ Cl₂ under N₂ at0° C. was treated with dimethyldioxirane (0.09M, 80 mL) and stired untilall of the glycal was consumed (0.5-1 h; TLC 30% EtOAc in hexanes).Volatiles were removed at 0° C. with stream of dry N₂. The residue wasdissolved in 30 mL of dry THF under N₂ at 0° C. and treated TBAF (36 mL,stored over molecular sieves) then stirred at ambient temperature for 20h. The dark brown solution was filtered through a pad of silica (˜4 cmdepth) and washed with EtOAc (200 mL). The filtrate was washed withwater (200 mL×3) and dried (MgSO₄) and concentrated. The residue wasredissolved in 30% EtOAc-hexanes (50 mL) and filtered through shortsilica column (10 cm d×4 cm h) and washed with the same solvent system(1 L). The filtrate was concentrated to give 2.59 g of fluorohydrinwith >90% purity. The residue was dissolved in dry DMF (30 mL) under N₂at 0° C. and treated with benzyl bromide (958 uL, 1.5 equiv, freshlyfiltered through basic alumina), finally with NaH (322 mg, 60%dispersion, 1.5 equiv) and stirred for 30 min at 0° C. and 30 min at rt.The reaction was quenched by pouring into 100 g of ice, and extractedwith 1:1 EtOAc-hexanes (150 mL×2). The organic extracts were washed withwater (150 mL×2), dried (MgSO₄) and concentrated in vacuo. Flashchromatography with 10% EtOAc-hexanes gave 2.00 g (49%) of the titlecompound as a yellowish liquid.

[α]²³ _(D) =+15.3° (CHCl₃, c=0.85); IR (CHCl₃ film) 2916, 1612, 1513,1248, 1103, 1056, 734 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃) δ 7.35-7.24 (17H,m), 6.84 (2H, d, J=8.4 Hz), 5.15 (1H, dd, J=53.2, 7.0 Hz), 4.92 (lHz, d,J=11.6 Hz), 4.48-4.74 (2H, ABq, J=11.8 Hz), 3.96-3.89 (1H, m), 3.86 (1H,br s), (3H, s), 3.65-3.56 (3H, m), 3.51 (1H, dd, J=9.8, 2.8Hz); ¹³ C-NMR(100 MHz, CDCl₃) δ 159.22, 138.33, 138.11, 137.62, 130.16, 129.19,128.40, 128.29, 128.21, 128.04 (2C), 127.90, 127.81, 127.69, 127.59,113.77, 110.20 (d, J=214 Hz), 80.60 (d, J=11.3Hz), 79.00 (d, J=20.5 Hz),74.92, 74.52, 73.59 (d, J=5.0Hz), 73.54, 72.99, 72.70, 68.34, 55.20;LRMS (NH₃) 454 (M+NH₄ ⁺, 100).

Synthesis of 6b:

A solution of TIPS-carbonate galactal 5b (Danishefsky, S. J.; Behar, V.;Randolph, J. T.; Lloyd, K., J. Am. Chem. Soc., 1995, 5701)(4.28 g, 5.62mmol) in THF (25 mL)-MeOH (5 mL) was treated with TBAF solution (1.0M,6.75 mL, 1.2 equiv). After 6 h, additional TBAF (4 mL) was added andstirred additional 3 h. The reaction was concentrated and directlychromatographed with 4:1 EtOAc-hexanes to obtain 2.20 g of the triol.Remaining mixtures of cyclic carbonate and mixed carbonate washydrolysed in MeOH with MeONa (1.0 mL, 25 wt %) and purifiedchromatographically. Total yield was 3.02 g (93%). This material wasdirectly used for the dibenzylation step.

¹ H-NMR (400 MHz, CDCl₃) δ 7.35-7.24 (15H, m), 6.43 (1H, d, J=6.3 Hz),4.87 (1H, dd, J=6.3, 3.4 Hz), 4.84 (1H, d, J=11.4 Hz), 4.63 (2H,apparent s), 4.61 (1H, d, J=11.4 Hz), 4.53-4.47 (3H, m), 4.19-4.16 (3H,m), 3.87-3.84 (2H,m), 3.78-3.66 (3H, m), 3.46 (2H, apparent d, J=4.6Hz), 3.29 (1H, t, J=5.5 Hz), 3.08 (1H, br), 2.73 (2H, br); ¹³ C-NMR (100MHz, CDCl₃) δ 144.70, 138.41, 138.22, 137.83, 128.45, 128.33 (2C),128.12, 127.84, 127.73, 127.64, 127.57, 102.28, 99.74, 78.99, 76.03,74.64, 74.07, 73.24 (2C), 73.17, 72.64, 70.20, 69.10, 67.79, 62.15.

A mixture of triol glycal from above (2.95 g, 5.1 mmol), dibutyltinoxide (1.33 g, 1.05 equiv) and bistributyltin oxide (1.69 mL, 0.65equiv) in dry benzene (50 mL) under N₂ was refluxed for 5 h withazeotropic removal of water. The reaction was cooled below boiling andtreated with benzyl bromide (2.43 mL, 4.0 mol equiv) andtetrabutylammonium bromide (3.29 g, 2.0 equiv). 10 mL of benzene wasdistilled off and the reaction refluxed for 16 h. The reaction wasdirectly loaded on silica column and eluted with 15-20% EtOAc-hexanes togive 3.48 g (90%) of product 6b as a clear oil.

[α]²³ _(D) =-3.3° (CHCl₃, c=0.87); IR (CHCI₃ film) 2867, 1652 1454,1364, 1097, 736 cm⁻¹ ; ¹ H-MNR (400 MHz, CDCl₃) δ 7.35-7.21 (25H, m),6.45 (1H, d, J=6.2 Hz), 4.88 (1H, dd, J=6.2, 3.9 Hz), 4.83 (1H, d,J=10.9 Hz), 4.69 (2H, apparent s), 4.68 (1H, d, J=10.9 Hz), 4.59 (2H,apparent s), 4.55 (1H, d, J=7.8 Hz), 4.49 (2H, apparent s), 4.47 (2H,apparent s), 4.29 (1H, dd, J=9.6, 5.8 Hz), 4.18 (1H, t, J=4.4 Hz), 4.13(1H, m), 3.99 (1H, br s), 3.85 (1H, dd, J=10.6, 6.4 Hz), 3.75-3.60 (4H,m), 3.47-3.41 (2H, m); ¹³ C-NMR (100 MHz, CDCl₃) δ 144.43, 138.64,138.42, 137.99, 137.84, 137.80, 128.40, 128.34, 128.26, 128.23, 128.18,128.15, 127.82, 127.75, 127.69, 127.67, 127.65, 127.55, 127.51, 127.46,127.31, 102.56, 99.56, 80.57, 78.69, 75.72, 75.10, 73.57, 73.32, 72.94,72.28, 71.94, 70.12, 68.90, 67.85, 66.62; LRMS (NH₃) 776 (M+NH₄ ⁺, 100).

Synthesis of 7b:

Lactal 6b (1.32 g, 1.74 mmol, 1.0 equiv) and fluoro sugar 4b (1.49 g,2.60 mmol, 1.5 equiv) were combined in ether and concentrated. Themixture were dried by evaporation in dry benzene (25 mL×2), in vacuumfor 2 h then treated with di-t-butylpyridine (389 uL, 1.0 equiv) inglove bag and dissolved in dry ether (18 mL) under nitrogen atmosphere.In a separate 50 mL flask was placed 4A M.S. (4.0 g) then flame-driedunder vacuum, cooled to room temperature. Anhydrous silver perchlorate(360 mg, 1.0 equiv) and SnCl₂ (329 mg, 1.0 equiv) were added in glovebag and flushed with nitrogen. The salt mixture was placed in water bathand sugar solution was introduced via double tipped needle and thedixture sonicated for 2 min. The reaction was wrapped with aluminum foiland stirred for 45 h at rt. The filtrate (200 mL) was washed with dilNaHCO3 (100 mL×2), dried (MgSO₄) and concentrated. Flash chromatographywith 15-20% EtOAc/hexanes yielded trisaccharides (1.107 g, 49%) andimpure lactal. The trisaccharide portion was rechromatographed with 2%ether-methylene chloride to give 879 mg (39%) of the desired α-productand 195 mg (8.6%) of β-product. The impure lactal fraction wasrechromatographed with 3-4% ether-methylene chloride to give 479 mg(36%) of clean lactal. 77% of coupling (61% α-product) yield based onrecovered starting material.

[α]²³ _(D) =+41.8° (CHCl₃, c=1.8); IR (CHCl₃ film) 2867, 1648, 1513,1496, 1453, 1364, 1248, 1097, 735 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃) δ7.33-7.12 (42H, m) 6.83 (2H, d, J=8.4 Hz), 6.45 (1H, d, J=6.0 Hz), 5.03(1H, d, J=2.3 Hz), 4.91-4.76 (6H, m), 4.68-4.40 (12H, m), 4.23-3.97(11H, m), 3.86-3.82 (1H, dd, J=2.3 Hz), 3.76 (3H, s), 3.69-3.64 (2H, m),3.53 (1H, t, J=8.7 Hz), 3.47-3.43 (1H, m), 3.40-3.36 (1H, m), 3.34-3.31(1H, dd, J=9.9, 2.8 Hz), 3.22 (1H, dd, J=8.3, 4.8 Hz); ¹³ C-NMR (100MHz, CDCl₃) δ 158.93, 144.59, 138.98, 138.84, 138.78, 138.64, 138.58,138.06, 138.02 (2C), 130.82, 129.04, 128.33, 128.24, 128.21, 128.15,128.08, 128.05, 127.83, 127.81, 127.72, 127.64, 127.58, 127.55, 127.50,127.44, 127.41, 127.36, 127.33, 127.31, 113.65, 103.02, 100.39, 100.01,80.93, 78.93, 78.70, 76.53, 76.11, 75.14, 74.84, 74.79, 74.35, 73.91,73.59, 73.36, 73.15, 73.10, 72.98, 72.15, 72.10, 71.99, 70.55, 69.25,67.92 (2C), 67.69, 55.19.

Synthesis of 8b:

A solution of PMB-trisaccharide (37 mg, 0.028 mmol) in CH₂ Cl₂ (1 mL) at0° C. The reaction was directly loaded on silica column and eluted with20% EtOAc-hexanes to give 28 mg (84%) of desired product.

[α]²³ _(D) =+45.6° (CHCl₃, c=1.78); IR (CHCl₃ film) 2866, 1648, 1496,1453, 1248, 1097, 735 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃) δ 7.36-7.15 (40H,M), 6.43 (1H, d, J=6.2 Hz), 5.09 (1H, d, J=3.3 Hz), 4.85 (1H, dd, J=6.2,3.6 Hz), 4.83-4.65 (5H, m), 4.61-4.41 (9H, m), 4.29-4.08 (8H, m), 4.02(1H, d, J=2.6 Hz), 3.97 (1H, d, J=2.2 Hz), 3.93 (1H, t, J=8.4 Hz),3.86-3.78 (2H, m), 3.67-3.61 (2H, m), 3.53 (1H, dd, J=8.5, 4.8 Hz); ¹³C-NMR (100 MHz, CDCl₃) δ 144.38, 138.78, 138.62, 138.47, (2C), 138.20,138.00, 137.88, (2C, 128.31, 128.29, 128.23, 128.19, 128.16, 128.05,127.88, 127.83, 127.62, 127.57, 127.49, 127.45, 127.43, 127.41, 127.37,127.32, 127.23, 102.68, 99.89, 99.34, 80.82, 78.72, 77.49, 77.10, 75.88,75.13, 75.03, 74.23, 73.62, 73.05, 73.01, (3C), 72.62, 72.19 (2C),70.46, 69.66, 68.92, 67.85, 67.74, 67.54.

Synthesis of 11b:

Glycal 9b (4.32 g, 3.14 mmol) was dissolved in CH₂ Cl₂ (20 ml) andcooled to 0° C. It was then treated with 3,3-dimethyldioxarane (219 ml,˜3.14 mmol) at 0° C. The epoxidation finished within 1 h and then thereaction mixture was concentrated to dryness using dry N₂ stream. Theresidue was further azeotroped once with benzene (20 ml) and put on avacuum line for 30 min at 0° C. before being dissolved in THF (60 ml)and cooled to -78° C. Into the above solution was added, via canula,azeotropically dried galactal 10b (3.32 g, 10.95 mmol, 20 ml THF) andfollowed by ZnCl₂ (26.3 ml, 1.0 M in ether). The reaction mixture waswarmed up to room temperature and stirred overnight. After treatmentwith sat'd aq. Na₂ CO₃ (40 ml), the reaction mixture was concentratedand extracted with ether (500 ml). The organic phase was washed withsat'd aq. NaCl, dried (MgSO₄) and concentrated. The crude product waspurified by silica gel chromatography (1:4 EtOAc-hexanes) to give 6.20 gof 11b as a white foam (87.4%).

IR (CHCl₃ film) xyz cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃) δ 6.45 (1H, dd,J=6.4, 1.6 Hz), 4,85 (1H, dd, J=6.4, 2.0 Hz), 4.72-4.68 (2H, m), 4.65(1H, d, J=7.2 Hz), 4.55 (1H, m), 4.21 (1H, m), 4.08 (1H, dd, J=9.6, 5.6Hz), 3.96-3.82(6H, m), 3.33 (1H, d, J=3.2 Hz, OH), 3.27 (1H, d, J=2.8Hz, OH), 1.16-1.04 (42H, m); ¹³ C-NMR (100 MHz, CDCl₃) δ 154.45, 145.75,99.14, 98.27, 77.83, 76.59, 74.27, 72.04, 71.62, 70.86, 64.52, 62.57,61.60, 17.84, 11.78, 11.77.; LRMS (NH₃) 664 (M+NH₄ ⁺, 100), 647 (M+1⁺,5), 422 (21), 380 (25).

Synthesis of 13b:

Disaccharide 11b (2.64 g, 4.08 mmol) was azeotropically dried threetimes (3×10 ml) together with fluoro-fucose 12b (1.64 g, 3.77 mmol) andmolecular sieves (4 A, 4.0 g) in THF (20 ml) with2,6-di-tert-butylpyridine. The solution was added via canula to a flaskcontaining AgClO₄ (1.56 g, 7.54 mmol), SnCl₂ (1.43 g, 7.54 mmol) andmolecular sieves (4 A, 4.0 g) in THF (15 ml) at -40° C. The reactionmixture was stirred 30 min at -40° C. and then 34 h at 5° C. until thedisappearance of fluoro-fucose. After treatment with sat'd aq. NaHCO₃(40 ml) at 5° C., the mixture was extracted with EtOAc (700 ml). Theorganic phase was washed with sat'd NaCl, dried (MgSO₄) andconcentrated. The crude product was purified by silica gelchromatography to give 1.93 g of the desired trisaccharide glycal 13b(48%, based on fluoro-fucose used) and 500 mg of the recovereddisaccharide with only a trace of the other monofucosyl product.

Synthesis of 15b:

An azeotropically dried mixture of the trisaccharide glycal 13b (1.11 g,1.05 mmol) and benzensulphonamide (0.82 g, 5.24 mmol) was dissolved inthe THF (20 ml) together with molecular shieves (4 A, 2.6 g). Themixture was cooled to -40° C. and then was added, via canula, a solutionof I(sym-coll)₂ ClO₄ prepared in situ by stirring I₂ (0.54 g, 2.09 mmol)with Ag(sym-coll)₂ ClO₄ (0.986 g, 2.29 mmol) in THF (20 ml) at roomtemperature for about 30 min until the disappearance of the brown colorof I₂. The mixture was warmed up to 0° C. within 1 h and stirred foranother 1 h. After quenching with sat'd aq. Na₂ S₂ O₃, the mixture wasfiltrate and extracted with EtOAc (3×100 ml). The combined organic phasewas washed with sat'd aq. CuSO4 (100 ml), sat'd NaCl (100 ml×2) anddried (Na₂ SO₄). After concentration, the crude product was purified bysilica gel chromatography (1:4 EtOAc-hexanes) to give 981 mg of acolorless oil as a 2:1 mixture of the desired α-trans-diaxialiodosulphonamide and its cis isomer. The iodosulphonamide mixture wasthen added with stirring into a flask containing ethanthiol (226.3 mg,3.64 mmol) and lithium hexamethydisilylazide (1.46 ml, 1.46 mmol) in DMF(10 ml) at -40° C. The reaction mixture was stirred at -40° C overnight,and then quenched with sat'd aq. NaHCO₃ and extracted with ether (3×100ml). The combined organic phase was washed with sat'd aq. NaCl and dried(Na₂ SO₄). After concentration, the crude product was purified by silicagel chromatography (3:97 EtOAc-CH₂ Cl₂) to yield 438 mg of 15b (33%) and333 mg of the intact cis iodosulphonamide.

Synthesis of 16b:

A mixture of acceptor trisaccharide 8b (92 mg, 0.077 mmol, 1.0 equiv),thioglycoside 15b (198 mg, 2.0 equiv) and freshly activated 4Å-MS (560mg) under N₂ at rt was suspended in CH₂ Cl₂ -Et₂ O (1:2, 3.9 mL) andstirred for 10 min. The reaction was cooled to 0° C., then treated withmethyl triflate (52.4 uL, 6.0 equiv). The reaction was stirred for 4.5 hat 0° C. and 1.5 h while warming to 15° C. The reaction was quenchedwith TEA(1.0 mL), filtered through a pad of silica and rinsed with Et₂O. The filtrate (70 mL) was washed with sat'd NaHCO₃ (50 mL×2), dried(Na₂ SO₄) and concentrated. The crude product was purified by HPLC (17%EtOAc in hexanes, 15 mL/min, 260 nm UV dection) to give 158 mg (85%) ofthe desired product and 27.7 mg of α-linked byproduct (ca 55% purity).

Retention time=22 min; [α]²³ _(D) =-13.30 (CHCl₃, c=1.4); IR (CHCl₃film) 2940, 2865, 1792, 1652, 1454, 1161, 1101, 734 cm⁻¹ ; ¹ H-NMR (400MHz, CDCl₃) δ 7.8 (2H, m), 7.38-7.06 (58H, m), 6.43 (1H, d, J=6.1 Hz),5.15 (1H, br s), 5.07 (1H, d, J=3.6 Hz), 5.03 (1H, d, J=3.6 Hz), 4.99(1H, d, J=11.6 Hz), 4.89-4.61 (12H, m), 4.54-4.46 (4H, m), 4.42 (2H, apps), 4.38 (1H, d, J=11.9 Hz), 4.34-4.26 (3H, m), 4.21-4.18 (4H, m),4.13-4.03 (7H, m), 3.98-3.76 (14H, m), 3.70-3.61 (4H, m), 3.46-3.27 (7H,m), 2.84 (1H, OH), 1.16 (3H, d, J=6.4 Hz), 1.13-1.02 (42H, m); ¹³ C-NMR(100 MHz, CDCl₃) δ 155.35, 144.55, 140.78, 138.99, 138.75, 138.68,138.59, 138.54, 138.43, 138.13, 138.03, 137.94, 137.82, 132.31, 128.81,128.52, 128.51, 128.38, 128.36, 128.27, 128.24, 128.20, 128.16, 128.02,127.93, 127.72, 127.66, 127.58, 127.48, 127.43, 127.37, 127.20, 103.41,102.75, 99.79, 99.55, 98.29, 97.76, 80.49, 80.39, 79.09, 78.91, 78.25,77.68, 77.37, 76.51, 75.88, 75.09, 74.99, 74.91, 74.73, 74.15, 74.02,73.92, 73.52, 73.19, 73.10, 72.94, 72.67, 72.25, 72.07, 71.76, 71.56,71.33, 70.33, 69.45, 69.32, 68.48, 68.08, 67.86, 67.75, 61.97, 61.60,56.14, 17.99, 17.96, 17.95, 17.92, 16.75, 11.86; HRMS (FAB) calcd forC₁₃₈ H₁₆₉ NO₃₀ SSi₂ Na (M+Na) 2432.0920, found 2432.0970.

Synthesis of 19b:

A solution of hexasaccharide glycal 16b (85 mg, 0.035 mmol) in THF (6mL) under N₂ at rt was treated with TBAF (1.0 M, 353 uL, 10 equiv).After 38 h at rt, the reaction was concentrated to ca 1 mL, thendissolved in EtOAc (60 mL), washed with water (30 mL×2), dried (Na₂ SO₄)and concentrated. Flash chromatography with 4% MeOH in CH₂ Cl₂ gave 70.0mg (98%) of the desilyl-decarbonated product.

[α]²³ _(D) =1.8° (CHCl₃ film) 2868, 1652, 1455, 1157, 1094, 735 cm⁻¹ ; ¹H-NMR (400 MHz, CDCl₃) δ 7.80 (2H, d, J=7.4 Hz), 7.47 (2H, d, J=7.2 Hz),7.37-6.95 (56H, m), 6.45 (1H, d, J=6.3 Hz), 5.86 (1H, br s), 5.35 (1H,d, J=11.6 Hz), 5.30 (1H, D, J=2.8 Hz), 4.95 (1H, d, J=11.3 Hz), 4.89(1H, d, J=3.5 Hz), 4.8644.67 (9H, m), 4.54-4.39 (9H, m), 4.34 (1H, dd,J=10.4, 2.8 Hz), 4.26-4.06 (9H, m), 3.98-3.45 (23H, m), 3.41 (1H, d,J=10.0 Hz), 3.29-3.20 (5H, m), 0.73 (3H, d, J=6.3 Hz); ¹³ C-NMR (100MHz, CDCl₃) δ 144.87, 142.49, 139.49, 139.11, 138.87, 138.63, 138.54,138.37, 138.00, 137.98, 137.97, 137.18, 131.64, 128.74, 128.52, 128.43,128.33, 128.28, 128.25, 128.21, 128.02, 127.99, 127.97, 127.80, 127074,127.67, 127.63, 127.61, 127.54, 127.53, 127.50, 127.44, 127.33, 127.31,127.02, 126.86, 103.39, 102.78, 100.75, 100.09, 99.80, 99.75, 81.42,80.64, 78.98, 78.86, 77.82, 77.40, 77.26, 76.26, 75.16, 75.09, 75.07,74.95, 74.69, 74.30, 73.58, 73.17, 73.11, 72.71, 72.67, 72.65, 72.55,72.36, 72.18, 69.65, 69.53, 68.54, 68.18, 68.08, 67.85, 67.79, 67.21,54.95, 16.60.

To liquid ammonia (ca 8 mL) under N₂ at -78° C. was added metalic sodium(95 mg) and stirred for 2 min. To the blue solution was added a solutionof th hexasaccharide glycal above (70 gm, 33.8 umol) in dry THF(2 mL).After 45 min at 78° C., the reaction was quenched with absolute methanol(4 mL). Most of ammonia was removed with stream of nitrogen (finalvolume was ca 4 mL) and the reaction diluted with methanol to ca 10 mL.To the solution was added Dowex 50-X8 (890 mg, washed and dried) andstirred for 5 min. The solution was filterate and rinsed with methanol,finally with ammoniacal methanol (5 mL), and the filterate wasconcentrated in vacuo. The residue and DMAP (2.4 mg) were placed underN₂ and suspended in DMF (1.0 mL), THF (1.0 mL) and TEA (1.0 mL), thentreated with Ac₂ O (0.3 mL). After 20 h (TLC analysis with EtOAc), thereaction was poured into water (40 mL), and extracted with EtOAc (40mL×2), washed with dil NaHCO₃ (30 mL), with water (30 mL), dried (Na₂SO₄) and concentrated. Flash chromatography with 80% EtOAc in CH₂ Cl₂gave 52.0 mg (93%) of product as white foam.

mp 132-134° C.; [α]²³ _(D) =+4.7° (CHCl₃, c=1.4); IR (CHCl₃ film) 1742,1652, 1371, 1227, 1069 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃) δ 6.68 (1H, d,J=6.8 Hz), 6.42 (1H, d, J=6.0 Hz), 5.58 (1H, d, J=3.2 Hz), 5.47 (1H, d,J=3.4 Hz), 5.40-5.37 (2H, m), 5.29 (1h, dd, J=10.9, 3.1 Hz), 5.25-5.15(5H, m) 5.06 (1H, dd, J=11.2, 3.3 Hz), 5.02 (1H, d, J=3.6 Hz), 4.99-4.92(2H, m), 4.84-4.81 (2H, m), 4.67 (1H, d, J=7.8 Hz), 4.56-4.51 (2H, m),4.45-4.38 (3H,m), 4.29 (1H, dd, J=10.6, 3.4 Hz), 4.22-3.95 (13H, m₋₋,3.90-3.77 (3H, m), 2.19-1.92 (51H, m), 1.15 (3H, d, J=6.4 Hz); ¹³ C-NMR(100 MHz, CDCl₃) δ 172.40, 171.45, 170.84, 170.54, 170.52, 170.48,170.45, 170.40, 170.39, 170.34, 170.23, 169.99, 169.82, 169.74, 169.36,169.00, 145.43, 102.01, 101.17, 98.83, (2C), 98.45, 94.24, 75.65, 74.95,73.98, 73.64, 73.49, 72.32., 71.84, 71.53, 71.44, 70.81, 70.74, 70.66,70.12, 69.77, 68.97, 68.71, 68.67, 68.02, 67.97, 67.88, 67.60, 67.35,64.43, 61.88, 61.81, 61.42, 61.29, 61.04, 56.18, 23.06, 21.02, 20.81,20.76, 20.68, 20.64, 20.62, 20.58, 20.57, 20.55, 20.49, 20.43, 15.88.

Peracetyl hexasaccharide glycal above (52 mg) was divided into twoportions (22 mg and 30 mg). A solution of hexasaccharide glycal (22.0mg, 13.4 umol) in dry CH₂ Cl₂ (2 mL) under N₂ at 0° C., then treatedwith allyl alcohol (5 mL). The mixture was stirred for 15 h at roomtemperature. Excess allyl alcohol was removed in vacuo. The other batch(30 mg) was treated similarly. The crude products were combined andchromatographed with 85% EtOAc-CH₂ Cl₂ to give 35.8 mg (66%) of lesspolar product and 15.7 mg (29%) of more polar product. A 33.2 mg (19umol) of the less polar material under N₂ was dissolved in absolute MeOH(14 mL) and treated with MeONa solution in methanol (165 uL, 25% byweight). After 6 h, the reaction was neutralized with Dowex 50-X8 (200mg, washed and dried), filtered and concentrated to give quantitativeyield of the title compound 19b.

mp 204-206° (dec); [α]²³ _(D) =+5.5° (MeOH, c=0.67); IR (MeOH film) 3356(br), 2923, 1658, 1374, 1071 cm⁻¹ ; ¹ H-NMR (400 MHz, CD₃ OD) δ5.99-5.93 (1H, m), 5.24 (1H, d, J=3.8 Hz), 5.18-5.14 (1H, m), 4.93 (1H,d, J=3.9 Hz), 4.56-4.54 (2H, m), 4.42-4.06 (10H, m), 3.99 (1H, s),3.91-3.47 (26H, m), 3.41-3.37 (1H, m), 3.27 (1H, t, J=8.8 Hz), 2.01 (3H,s), 1.24 (3H, d, J=6.5 Hz); ¹³ C-NMR (100 MHz, CD₃ OD, ref=δ49.05) δ174.55, 135.73, 117.57, 105.48, 105.42, 103.94, 103.26, 102.79, 101.08,81.21, 80.67, 80.05, 79.20, 78.09, 76.79, 76.56, 76.48, 76.44, 76.41,75.54, 74.86, 74.68, 73.57, 72.63, 72.50, 71.57, 71.16, 70.64, 70.41,69.68, 68.16, 62.67, 62.64, 62.57, 61.96, 61.63, 53.11, 23.58, 16.78.

For the purposes of the preparative synthesis of structure 1b a ceramideprecursor was attached to the ABC trisaccharide (Scheme 5). Expoxidationof 7b, followed by reaction with the ceramide precursor 17b (as itstributylstannyl ether) promoted by Zn(OTF)₂ provided 20b. Acetylationand PMB removal proceeded smoothly to furnish 21b which is poised forcoupling with a suitable DEF trisaccharide donor.

When trisaccharide 15b was treated with MeOTf in the presence ofacceptor 21b, a 4:1 mixture of hexasaccharide isomers was obtained. Themajor product 22b was obtained in 50% yield.

The ceramide side-chain was elaborated by reduction of the azidefunctionality using Lindlar's catalyst under an atmosphere H₂ in thepresence of palmitic anhydride to provide 18b directly. Desilylation wasfollowed by dissolving metal deprotection of the sulfonamide and benzylgroups and MeOH quench to remove the carbonate and acetate groups.Peracetylation of the crude mixture afforded a 78% yield ofperacetylated hexasaccharide. Saponification of this material usingNaOMe provided the natural product 1b in 96% yield. The couplingconstants and chemical shifts of the anomeric protons of 1b matchedreported data. In addition, the product was characterized by exact mass,and ¹ H and ¹³ C NMR.

Synthesis of 20b:

The benzylated ceramide precursor (475 mg, 1.14 mmol) was dissolved in 4mL PhH. Bis(tribuyltin) ether (0.29 mL, 0.34 g, 0.57 mmol) was added andthe reaction vessel (equipped with a Dean-Stark trap) was heated toreflux. After 3 h the reaction was allowed to cool and was concentratedunder a flow of N₂. In a separate flask, the glycal 7b was dissolved in1 mL anhydrous CH₂ Cl₂ and the resulting solution was cooled to 0° C.and a solution of 3,3-dimethyldioxirane (2.8 mL, 0.25 mmol, 0.09 M inacetone) was added. After 45 min the solution was concentrated under aflow of N₂, then under vacuum. The tin ether was dissolved in 1 mLanhydrous THF and added via cannula to a mixture of Zn(OTf)₂ (170 mg,0.468 mmol) in 1 mL THF at -78° C. (wash 1×0.5 mL THF). The reaction wasallowed to warm to room temperature over 12 h and then was quenched withdistilled water. The aqueous phase was extracted 3× with EtOAc. Thecombined organic phases were dried over anhydrous MgSO₄. Flash columnchromatography (3:1 hexane/EtOAc, 3×16 cm silica gel) afforded 265 mg(66%) of the target compound 20b.

¹ H NMR (CDCl₃) δ 7.43-7.15 (m, 45H), 7.03 (d, J=8.6 Hz, 2H), 6.79 (d,J=8.6 Hz, 2H), 5.76 (dt, J=6.7, 15.4 Hz, 1H), 5.43 (dd, J=8.5, 15.4 Hz,1H), 5.07 (d, J=3.5 Hz, 1H), 5.05 (d, J=12.0 Hz, 1H), 4.90 (d, J=12.9Hz, 2H), 4.83-4.77 (m, 3H), 4.69 (d, J=12.0 Hz, 1H), 4.61 (d, J=11.9 Hz,1H), 4.54-4.45 (m, 3H), 4.42-4.25 (m, 7H), 4.18-4.05 (m, 6H), 4.01-3.91(m, 4H), 3.83 (dd, J=4.4, 10.6 Hz, 1H), 3.79 (s, 3H), 3.71-3.65 (m, 4H),3.57-3.32 (m,7H), 3.20 (m, 1H), 2.29 (bs, 1H), 2.11 (bq, J=6.7 Hz, 2H),1.42-1.29 (m, 22H), 0.91 (t, J=6.6 Hz, 3H); ¹³ C NMR (CDCl3) δ 158.8,139.1, 139.0, 138.7, 138.6, 138.34, 138.29, 138.2, 138.1, 130.8, 128.7,128.55, 128.50, 128.4, 128.33, 128.28, 128.26, 128.12, 128.06, 127.84,127.76, 127.7, 127.64, 127.60, 127.5, 127.45, 127.36, 125.8, 113.5,102.7, 100.6, 81.9, 81.5, 79.4, 77.4, 77.0, 76.7, 76.6, 76.4, 75.5,74.9, 74.7, 74.4, 73.9, 73.3, 73.2, 73.11, 73.06, 72.3, 72.1, 70.0,69.4, 68.7, 68.1, 67.9, 67.7, 64.2, 55.2, 32.4, 31.9, 29.70, 29.65,29.5, 29.4, 29.2, 29.0, 22.7, 14.2; IR (thin film) 3447, 3062, 3029,2923, 2853, 2099, 1612, 1586, 1514, 1496, 1454, 1364 cm⁻¹ ; [α]²³ _(D)+25.0 (c 0.70).

Synthesis of 21b:

The above trisaccharide (256 mg, 0.147 mmol) was dissolved in 2 mLanhydrous CH₂ Cl₂. Triethylamine (0.105 mL, 76 mg, 0.753 mmol), DMAP (2mg, 0.02 mmol) and acetic anhydride (0.042 mL, 45 mg, 0.445 mmol) wereadded sequentially. The reaction was stirred for 1 h then quenched withsaturated aqueous NaHCO₃. The extracts were dried with anhydrous MgSO₄,filtered and concentrated. Purification by flash column chromatography(4:1 hexane/EtOAc, 2×16 cm silica gel) afforded 235 mg (90%) of thedesired compound.

¹ H NMR (CDCl₃) δ 7.42-7.17 (m, 45H), 7.03 (d, J=8.6 Hz, 2H), 6.81 (d,J=8.6 Hz, 2H), 5.75 (dt, J=6.7, 15.4 Hz, 1H), 5.43 (dd, J=8.6, 15.4 Hz,1H), 5.07 (d, J=3.4, 1H), 4.99-4.90 (m, 4H), 4.85 (d, J=11.3 Hz, 2H),4.77 (d, J=11.9 Hz, 1H), 4.76 (d, J=12.4 Hz, 1H), 4.70 (d, J=12.0 Hz,1H), 4.62 (d, J=11.7 Hz, 1H), 4.57-4.52 (m, 3H), 4.49-4.34 (m, 7H), 4.30(d, J=11.8 Hz, 1H), 4.25 (d, J=11.8 Hz, 1H), 4.14-4.06 (m, 7H),4.01-3.95 (m, 2H), 3.91 (dd, J=5.6, 8.6 Hz, 1H), 3.85 (dd, J=4.3, 11.1,Hz, 1H), 3.80 (s, 3H), 3.74 (d, J=9.8 Hz, 1H), 3.69 (dd, 7.7, 9.9 Hz,1H), 3.63-3.51 (m, 5H), 3.43-3.34 (m, 3H), 3.22 (dd, J=4.6, 8.2 Hz, 1H),2.12 (dd, J=6.8, 13.6, 2H), 1.87 (s, 3H), 1.43-1.30 (m, 22H), 0.93, (t,J=6.6 Hz, 3H); ¹³ C NMR (CDCl₃) δ 169.3, 158.8, 139.1, 139.0, 138.69,138.65, 138.6, 138.31, 138.26, 138.2, 138.1, 138.0, 130.8, 128.8, 128.6,128.41, 128.35, 128.30, 128.28, 128.14, 128.0, 127.9, 127.8, 127.64,127.60, 127.58, 127.51, 127.47, 127.38, 126.0, 113.5, 102.7, 100.8,1006, 81.5, 79.9, 79.5, 79.4, 79.3, 77.4, 77.1, 76.8, 75.5, 75.3, 74.9,74.5, 74.2, 73.9, 73.2, 73.1, 73.0, 72.4, 72.2, 72.1, 70.2, 69.4, 68.1,68.0, 67.9, 67.5, 63.8, 55.2, 32.4, 32.0, 29.72, 29.67, 29.5, 29.4,29.2, 29.1, 22.7, 20.9, 14.2; IR (thin film) 3028, 2923, 2852, 2098,1751, 1611, 1513, 1496, 1453, 1365, 1232 cm¹⁻ ; [α]²³ _(D) +20.3 (c0.45).

The trisaccharide from above (230 mg, 0.129 mmol) was dissolved in 4 mLCH₂ Cl₂. Distilled water (1 mL) was added and the mixture was cooled to0° C. DDQ (35 mg, 0.15 mmol) was added and the reaction was stirred for1 h. The reaction was quenched with saturated aqueous NaHCO₃. Theaqueous phase was extracted 3× with CH₂ Cl₂. The combined organic phaseswere washed dried over anhydrous MgSO₄. Flash column chromatography (4:1hexane/EtOAc, 2×16 cm silica) afforded 182 mg (85%) of the targetcompound 21b.

¹ H NMR (CDCl₃) δ 7.38-7.13 (m, 45H), 5.73 (dt, J=6.7, 15.4 Hz, 1H),5.41 (dd, J=8.6, 15.4 Hz, 1H), 5.09 (d, J=3.2 Hz, 1H), 4.98 (d, J=12.5Hz, 1H), 4.95 (dd, J=8.0, 9.2 Hz, 1H), 4.87 (d, J=11.2 Hz, 1H), 4.80 (d,J=11.3 Hz, 1H), 4.77 (d, J=10.9 Hz, 1H), 4.70 (d, J=11.4 Hz, 1H),4.65-4.50 (m, 6H), 4.45-4.42 (m, 3H), 4.38-4.34 (m, 3H), 4.28 (bs, 2H),4.15 (d, J=11.7 Hz, 1H), 4.11 (d, J=11.8 Hz, 1H), 4.08-4.01 (m, 3H,3.98-3.94 (m, 3H), 3.88 (dd, J=5.5, 8.5 Hz, 1H), 3.82 (dd, J=4.3, 7.0Hz, 1H), 3.77 (dd, J=3.1, 10.1 Hz, 1H), 3.70 (d, J=9.8 Hz, 1H),3.64-3.51 (m, 5H), 3.46 (dd, J=5.4, 9.4, 1H), 3.39 (m, 1H), 3.34-3.30(m, 2H), 3.21 (dd, J=4.7, 8.4 Hz, 1H), 2.09 (m, 2H), 1.90 (s, 3H), 1.84(d, J=5.1 Hz, 1H), 1.41-1.27 (m, 22H), 0.90 (t, J=6.5 Hz, 3H); ¹³ C NMR(CDCl3) δ 169.3, 165.9, 139.3, 138.7, 138.6, 138.5, 138.3, 138.2, 138.1,138.0, 128.5, 128.4, 128.32, 128.27, 128.25, 128.17, 128.00, 127.94,127.91, 127.8, 127.75, 127.70, 127.67, 127.61, 127.55, 127.49, 127.45,127.21, 125.9, 107.8, 102.6, 100.8, 99.4, 81.4, 80.6, 79.3, 77.5, 77.3,77.0, 76.9, 76.7, 75.5, 75.3, 75.2, 74.3, 73.2, 73.1, 73.0, 72.9, 72.3,72.1, 70.1, 70.0, 69.1, 68.1, 68.0, 67.8, 67.4, 63.8, 32.4, 31.9, 29.7,29.6, 29.5, 29.4, 29.2, 29.1, 22.7, 20.9, 14.1; IR (thin film) 3570,3087, 3062, 3029, 2924, 2853 2099, 1950, 1873, 1752, 1496, 1453, 1366,1231 cm⁻¹ ; [α]²³ _(D) +17.6 (c 1.40).

Synthesis Of 22b:

Thioglycoside 15b (188 mg, 0.151 mmol) and the acceptor 21b (125 mg,0.0751 mmol) were azeotropically dried with benzene twice. The mixturewas then dissolved in 2.6 mL anhydrous Et₂ O and 1.3 mL CH₂ Cl₂ and tothis solution was added 500 mg of 4 Å mol. sieves. This mixture wasstirred for 1 h and then was cooled to 0° C. and MeOTf (0.051 mL, 74 mg,0.45 mmol) was added. The reaction was stirred at 0° C. for 9 h.Triethylamine (1 mL) was then added and reaction was filtered through aplug of silica and washed with Et₂ O. The filtrate was washed withsaturated aqueous NaHCO₃ and dried over anhydrous MgSO₄. Purification bypreparative HPLC (85:15 hexane/EtOAc) afforded 108 mg (50%) of thetarget compound 22b. The b/a ratio of the reaction was 4:1.

¹ H NMR (CDCl₃) δ 7.75 (d, J=7.2 Hz, 2H), 7.46-7.05 (m, 63H), 5.75 (dt,J=6.8, 15.2 Hz, 1H0, 5.43 (dd, J=8.6, 15.5 Hz, 1H), 5.13 (m, 2H), 5.09(d, 3.6 Hz, 1H), 5.05 (d, J=11.6 Hz, 1H), 5.00 (d, J=11.5 Hz, 1H),4.94-4.86 (m, 5H), 4.83-4.65 (m, 14H), 4.59 (d, 11.7 Hz, 2H), 4.53-4.43(m, 6H), 4.39-4.31 (m, 4H), 4.23 (d, J=11.9 Hz, 1H), 4.18 (d, J=11.9 Hz,1H), 4.15-4.08 (m, 2H), 4.05-3.57 (m, 31H), 3.54 (d, J=9.1 Hz, 1H),3.49-3.45 (m, 2H), 3.38 (m, 1H), 3.31-3.23 (m, 3H), 2.92-2.89 (m, 2H),2.75 (bt, 6.0 H, H), 2.12 (bq, J=6.9 Hz, 2H), 1.85 (s, 3H), 1.20-1.09(m, 42H), 0.92 (t, J=6.6 Hz, 3H); ¹³ C NMR (CDCl3) δ 169.1, 165.9,155.5, 140.9, 139.2, 139.0, 138.8, 138.64, 138.59, 138.47, 138.43,138.3, 138.2, 138.10, 138.07, 138.0, 132.1, 129.1, 128.69, 128.65,128.56, 128.43, 128.40, 128.36, 128.35, 128.26, 128.17, 128.12, 128.08,127.97, 127.77, 127.66, 127.64, 127.60, 127.54, 127.49, 127.45, 127.41,127.3, 126.0, 103.0, 102.7, 100.8, 99.7, 99.2, 98.0, 81.2, 80.6, 79.5,79.2, 79.0, 78.3, 77.7, 76.8, 76.5, 75.5, 75.3, 75.1, 75.03, 74.97,74.91, 74.87, 74.0, 73.2, 73.10, 73.07, 72.98, 72.93, 72.6, 72.3, 72.1,72.0, 71.32, 71.25, 70.2, 69.4, 69.32, 69.25, 68.1, 67.9, 67.5, 68.3,62.1, 62.0, 56.1, 32.4, 31.9, 29.71, 29.68, 29.66, 29.48, 29.38, 29.2,29.1, 22.7, 20.7, 18.13, 18.11, 18.01, 17.98, 16.9, 14.2, 11.9; IR (thinfilm) 3344, 3030, 2924, 2864, 2101, 1789, 1754, 1496, 1453, 1366, 1232cm⁻¹.

Synthesis of 18b:

The hexasaccharide 22b (66 mg, 0.023 mmol) was dissolved in 1 mL EtOAc.Lindlar's catalyst (66 mg)was added followed by the addition of palmiticanhydride (23 mg, 0.046 mmol). The system was purged under vacuum andthen put under 1 atm of H₂. After 24 h the reaction was filtered througha plug of silica gel, washed with EtOAc, and concentrated. Purificationby preparative HPLC (4:1 hexane/EtOAc) afforded 64 mg (90%) of thedesired product 18b.

¹ H NMR (CDCl₃) δ 7.72 (d, J=7.2 Hz, 2H), 7.42-7.02 (m, 63H), 5.65 (d,J=9.1 Hz, 1H), 5.62 (dt, J=6.6, 15.3 Hz, 1H), 5.31 (dd, J=8.6, 15.3 Hz,1H), 5.10 (m, 2H), 5.05 (d, J=3.6 Hz, 1H), 5.02 (d, J=11.5 Hz, 1H), 4.96(d, J=11.4 Hz, 1H), 4.90-4.62 (m, 13H), 4.57-4.38 (m, 8H), 4.32-4.26 (m,3H), 4.21-4.07 (m, 9H), 4.01-3.41 (m, 31H), 3.30 (m, 1H), 3.23 (m, 3H),2.20 (m, 4H), 1.82 (s, 3H), 1.52 (bm, 2H), 1.32-1.19 (m, 53H), 1.15-1.08(m, 42H), 0.88 (t, J=6.8 Hz, 6H); IR (thin film) 3531, 3346, 3063, 3030,2924, 2854, 1790, 1748, 1674, 1496, 1454, 1365, 1236 cm⁻¹ ; [α]²³ _(D)-17.9 (c 0.65).

Synthesis of 1b:

The hexasaccharide from above (20 mg, 0.0065 mmol) was dissolved in 0.5mL THF. A solution of tetrabutylammonium fluoride (1.0 M in THF, 0.050mL, 0.050 mmol) was added and the reaction was stirred for 2 h. Thesolution was filtered through a plug of silica, washed with EtOAc andconcentrated. The residue was dissolved in 1 mL of anhydrous MeOH andNaOMe (10 mg, 0.19 mmol) was added. The reaction was stirred for 3 h,neutralized with 40 mg of Dowex-50 resin, filtered and concentrated.Purification by flash column chromatography (1.5×4 cm 10-40 u silicagel, 95:5 CH₂ Cl₂ /MeOH) afforded 16.5 mg (94%) of the desired compound.

¹ H NMR (CDCl₃) δ 7.78 (d, J=7.6 Hz, 2H), 7.46 (d, J=7.4 Hz, 2H),7.41-6.97 (m, 61H), 6.02 (d, J=9.1 Hz, 1H) 5.76 (bs, 1H), 5.67 (dt,J=6.6, 15.3 Hz, 1H), 5.37-5.30 (m, 2H), 5.19 (d, J=2.6 Hz, 1H), 4.96 (d,J=11.3 Hz, 1H), 4.93 (d, J=3.4 Hz, 1H), 4.90-4.83 (m, 3H), 4.78-4.66 (m,7H), 4.56 (d, J=11.1 Hz, 1H), 4.53 (d, J=10.2 Hz, 1H), 4.47-4.32 (m,5H), 4.28-4.06 (m, 14H), 4.01-3.13 (m, 36H), 2.73 (bt, 1H), 2.61 (bs,1H), 2.54 (bs, 1H), (2.05 (m, 4H), 1.50 (m, 2H), 1.38-1.23 (m, 46H),0.88 (t, J=6.6 Hz, 6H), 0.78 (d, 6.3 Hz, 3H); ¹³ C NMR (CDCl3) δ 173.4,142.4, 139.5, 139.0, 138.7, 138.5, 138.33, 138.26, 138.14, 138.09,137.9, 137.2, 137.1, 131.6, 129.0, 128.8, 128.54, 128.47, 128.37,128.32, 128.27, 128.22, 128.17, 128.14, 128.05, 127.99, 127.79, 127.73,127.68, 127.63, 127.59, 127.49, 127.46, 127.37, 127.32, 126.98, 126.58,104.1, 102.83, 102.76, 100.3, 100.2, 82.1, 81.5, 81.2, 79.6, 79.2, 79.0,78.0, 77.3, 77.0, 76.7, 75.6, 75.3, 75.1, 75.0, 74.8, 74.6, 73.5, 73.4,73.2, 73.0, 72.7, 72.6, 71.9, 70.1, 69.6, 68.5, 68.2, 68.0, 67.5, 62.4,61.9, 54.8, 52.3, 36.9, 32.3, 31.9, 29.71, 29.67, 29.54, 29.50, 29.43,29.37, 29.28, 29.20, 25.7, 22.7, 16.7, 14.1; IR (thin film) 3424, 3062,3023, 2923, 2852, 1641, 1530, 1496, 1453, 1362, 1325 cm⁻¹ ; [α]²³ _(D)-3.2 (c 0.83).

A flask was equipped with a dry ice condenser and was charged with 4 mLNH₃. Sodium (18 mg, 0.78 mol) was added and to the resulting bluesolution was added 29 mg of the above hexasaccharide (0.010 mmol). Thereaction was stirred at -78° C. for 45 min. Quench by the addition ofMeOH (3 mL). Nitrogen was blown over the solution to evaporate the NH₃.The reaction was neutralized with 170 mg of Dowex-50 resin, filtered andconcentrated. The resulting residue was dissolved in 1 mL of 4:1THF/DMF. Triethylamine (0.5 mL) was added followed by the addition ofDMAP (3 mg) and acetic anhydride (0.200 mL). After 2 h the reaction wasconcentrated in vacuo. Purification by flash column (1.5×5 cm 10-40 msilica, 9:1 EtoAc/hexane) afforded 18 mg (78%) of the peracetate. Asample of this hexasaccharide (15 mg, 0.0065 mmol) was dissolved in 0.5mL of anhydrous MeOH and a NaOMe solution (30% in MeOH, 0.010 mL, 0.05mmol) was added. The solution was stirred for 3 h, neutralized with 9 mgDowex-50 resin, filtered and concentrated. The residue was purified byflash column chromatography (1.5×4 cm C-18 reverse phase silica, MeOH)to afford 9.6 mg of the natural product 1. Spectral data agree withthose reported by Hakomori, et al.

Biological Results

The MBR1 hexasaccharide has been prepared in two forms, the natural "B"form and the unnatural "A" form as shown below.

The natural structure ("β") is:

    Fucα1→2Galβ1→3GalNAcβ1→3Galα1.fwdarw.4Galβ1→4Gluβ1→1Cer

The unnatural structure "α" is:

    Fucα1→2Galβ1→3GalNAcα1→3Galα1.fwdarw.4Galβ1→4Gluβ1→1Cer

Both have been linked to ceramide to facilitate testing forimmunological reactivity with monoclonal antibody (mAb) MBr1.

By Thin Layer Chromatography (TLC) the 2 preparations migrate as similarsingle bands. Immune TLC (see Ritter, G., et al., Cancer Res. 50,1403-10 (1990)) demonstrates that both forms react with the MBr1monoclonal antibody specifically but that the β-form reacts 10 timesmore strongly (comparable staining is seen with 1/10 the amount ofantigen). The high level of reactivity of the β structure with mAb MBr1was confirmed using flow cytometry inhibition assays. Reactivity of MAbMBr1 with breast cancer cell lines such as MCF-7 was 98% inhibited by 8μg/ml of the β linkage preparation but was only 6% inhibited by 8 μg ofthe α-linkage preparation. GD3 ganglioside (negative control) showed noinhibition at all.

What is claimed is:
 1. A compound having the structure: ##STR81## wherein n is an integer between about 0 and about
 9. 2. The compound of claim 1 conjugated to a protein carrier.
 3. A pharmaceutical composition comprising an effective amount of the compound of claim 2 and a pharmaceutically acceptable carrier.
 4. A pharmaceutical composition comprising an effective amount of the compound of claim 1 and a pharmaceutically acceptable carrier.
 5. A compound having the structure: ##STR82## wherein n is an integer between about 0 and about
 9. 6. The compound of claim 5 conjugated to a protein carrier.
 7. A pharmaceutical composition comprising an effective amount of the compound of claim 6 and a pharmaceutically acceptable carrier.
 8. A pharmaceutical composition comprising an effective amount of the compound of claim 5 and a pharmaceutically acceptable carrier.
 9. A compound having the structure: ##STR83## wherein n is an integer between about 0 and about
 9. 10. The compound of claim 9 conjugated to a protein carrier.
 11. A pharmaceutical composition comprising an effective amount of the compound of claim 10 and a pharmaceutically acceptable carrier.
 12. The compound of claim 10, wherein the protein carrier is bovine serum albumin.
 13. A pharmaceutical composition comprising an effective amount of the compound of claim 9 and a pharmaceutically acceptable carrier. 